SMART MODE:

NORMAL GENOMIC

• Butryn A, Stoehr G, Linke-Winnebeck C, Hopfner KP
• Serendipitous crystallization and structure determination of cyanase (CynS) from Serratia proteamaculans.
• Acta Crystallogr F Struct Biol Commun. 2015; 71: 471-6
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• Cyanate hydratase (CynS) catalyzes the decomposition of cyanate and bicarbonate into ammonia and carbon dioxide. Here, the serendipitous crystallization of CynS from Serratia proteamaculans (SpCynS) is reported. SpCynS was crystallized as an impurity and its identity was determined using mass-spectrometric analysis. The crystals belonged to space group P1 and diffracted to 2.1 A resolution. The overall structure of SpCynS is very similar to a previously determined structure of CynS from Escherichia coli. Density for a ligand bound to the SpCynS active site was observed, but could not be unambiguously identified. Additionally, glycerol molecules bound at the entry to the active site of the enzyme indicate conserved residues that might be important for the trafficking of substrates and products.

• Matsuo T, Kuramoto H, Kumazaki T, Mitsui Y, Takahashi T
• LIN54 harboring a mutation in CHC domain is localized to the cytoplasm and inhibits cell cycle progression.
• Cell Cycle. 2012; 11: 3227-36
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• The mammalian LIN complex (LINC) plays important roles in regulation of cell cycle genes. LIN54 is an essential core subunit of the LINC and has a DNA binding region (CHC domain), which consists of two cysteine-rich (CXC) domains separated by a short spacer. We generated various LIN54 mutants, such as CHC deletion mutant, and investigated their subcellular localizations and effects on cell cycle. Wild-type LIN54 was predominantly localized in the nucleus. We identified two nuclear localization signals (NLSs), both of which were required for nuclear localization of LIN54. Interestingly, deletion of one CXC domain resulted in an increased cytoplasmic localization. The cytoplasmic LIN54 mutant accumulated in the nucleus after leptomycin B treatment, suggesting CRM1-mediated nuclear export of LIN54. Point mutations (C525Y and C611Y) in conserved cysteine residues of CXC domain that abolish DNA binding activity also increased cytoplasmic localization. These data suggest that DNA binding activity of LIN54 is required for its nuclear retention. We also found that LIN54 (C525Y) and LIN54 (C611Y) inhibited cell cycle progression and led to abnormal nuclear morphology. Other CXC mutants also induced similar abnormalities in cell cycle progression. LIN54 (C525Y) led to a decreased expression of some G2/M genes, whose expressions are regulated by LINC. This cell cycle inhibition was partially restored by overexpression of wild-type LIN54. These results suggest that abnormal cellular localization of LIN54 may have effects on LINC activity.

• Kamennaya NA, Post AF
• Characterization of cyanate metabolism in marine Synechococcus and Prochlorococcus spp.
• Appl Environ Microbiol. 2011; 77: 291-301
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• Cyanobacteria of the genera Synechococcus and Prochlorococcus are the most abundant photosynthetic organisms on earth, occupying a key position at the base of marine food webs. The cynS gene that encodes cyanase was identified among bacterial, fungal, and plant sequences in public databases, and the gene was particularly prevalent among cyanobacteria, including numerous Prochlorococcus and Synechococcus strains. Phylogenetic analysis of cynS sequences retrieved from the Global Ocean Survey database identified >60% as belonging to unicellular marine cyanobacteria, suggesting an important role for cyanase in their nitrogen metabolism. We demonstrate here that marine cyanobacteria have a functionally active cyanase, the transcriptional regulation of which varies among strains and reflects the genomic context of cynS. In Prochlorococcus sp. strain MED4, cynS was presumably transcribed as part of the cynABDS operon, implying cyanase involvement in cyanate utilization. In Synechococcus sp. strain WH8102, expression was not related to nitrogen stress responses and here cyanase presumably serves in the detoxification of cyanate resulting from intracellular urea and/or carbamoyl phosphate decomposition. Lastly, we report on a cyanase activity encoded by cynH, a novel gene found in marine cyanobacteria only. The presence of dual cyanase genes in the genomes of seven marine Synechococcus strains and their respective roles in nitrogen metabolism remain to be clarified.

• Sijacic P, Wang W, Liu Z
• Recessive antimorphic alleles overcome functionally redundant loci to reveal TSO1 function in Arabidopsis flowers and meristems.
• PLoS Genet. 2011; 7: 1002352-1002352
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• Arabidopsis TSO1 encodes a protein with conserved CXC domains known to bind DNA and is homologous to animal proteins that function in chromatin complexes. tso1 mutants fall into two classes due to their distinct phenotypes. Class I, represented by two different missense mutations in the CXC domain, leads to failure in floral organ development, sterility, and fasciated inflorescence meristems. Class II, represented by a nonsense mutation and a T-DNA insertion line, develops wild-type-like flowers and inflorescences but shows severely reduced fertility. The phenotypic variability of tso1 alleles presents challenges in determining the true function of TSO1. In this study, we use artificial microRNA, double mutant analysis, and bimolecular fluorescence complementation assay to investigate the molecular basis underlying these two distinct classes of phenotypes. We show that the class I mutants could be converted into class II by artificial microRNA knockdown of the tso1 mutant transcript, suggesting that class I alleles produce antimorphic mutant proteins that interfere with functionally redundant loci. We identified one such redundant factor coded by the closely related TSO1 homolog SOL2. We show that the class I phenotype can be mimicked by knocking out both TSO1 and its homolog SOL2 in double mutants. Such antimorphic alleles targeting redundant factors are likely prevalent in Arabidopsis and maybe common in organisms with many sets of paralogous genes such as human. Our data challenge the conventional view that recessive alleles are always hypomorphic or null and that antimorphic alleles are always dominant. This study shows that recessive alleles can also be antimorphic and can produce a phenotype more severe than null by interfering with the function of related loci. This finding adds a new paradigm to classical genetic concepts, with important implications for future genetic studies both in basic research as well as in agriculture and medicine.

• Barauna RA et al.
• Proteomics Analysis of the Effects of Cyanate on Chromobacterium violaceum Metabolism.
• Genes (Basel). 2011; 2: 736-47
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• Chromobacterium violaceum is a gram-negative betaproteobacterium that has been isolated from various Brazilian ecosystems. Its genome contains the cyn operon, which gives it the ability to metabolize highly toxic cyanate into ammonium and carbon dioxide. We used a proteomics approach to investigate the effects of cyanate on the metabolism of this bacterium. The proteome of cells grown with and without cyanate was compared on 2-D gels. Differential spots were digested and identified by mass spectrometry. The bacterium was able to grow at concentrations of up to 1 mM cyanate. Eighteen spots were differentially expressed in the presence of cyanate, of which 16 were downregulated and only two were upregulated. An additional 12 spots were detected only in extracts of cells unexposed to cyanate, and one was expressed only by the exposed cells. Fourteen spots were identified, corresponding to 13 different proteins. We conclude that cyanate promotes expression of enzymes that combat oxidative stress and represses enzymes of the citric acid cycle, strongly affecting the energetic metabolism of the cell. Other proteins that were under-expressed in bacteria exposed to cyanate are involved in amino-acid metabolism or are hypothetical proteins, demonstrating that cyanate also affects expression of genes that are not part of the cyn operon.

• Desai KK, Miller BG
• Recruitment of genes and enzymes conferring resistance to the nonnatural toxin bromoacetate.
• Proc Natl Acad Sci U S A. 2010; 107: 17968-73
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• Microbial niches contain toxic chemicals capable of forcing organisms into periods of intense natural selection to afford survival. Elucidating the mechanisms by which microbes evade environmental threats has direct relevance for understanding and combating the rise of antibiotic resistance. In this study we used a toxic small-molecule, bromoacetate, to model the selective pressures imposed by antibiotics and anthropogenic toxins. We report the results of genetic selection experiments that identify nine genes from Escherichia coli whose overexpression affords survival in the presence of a normally lethal concentration of bromoacetate. Eight of these genes encode putative transporters or transmembrane proteins, while one encodes the essential peptidoglycan biosynthetic enzyme, UDP-N-acetylglucosamine enolpyruvoyl transferase (MurA). Biochemical studies demonstrate that the primary physiological target of bromoacetate is MurA, which becomes irreversibly inactivated via alkylation of a critical active-site cysteine. We also screened a comprehensive library of E. coli single-gene deletion mutants and identified 63 strains displaying increased susceptibility to bromoacetate. One hypersensitive bacterium lacks yliJ, a gene encoding a predicted glutathione transferase. Herein, YliJ is shown to catalyze the glutathione-dependent dehalogenation of bromoacetate with a k(cat)/K(m) value of 5.4 x 10(3) M(-1) s(-1). YliJ displays exceptional substrate specificity and produces a rate enhancement exceeding 5 orders of magnitude, remarkable characteristics for reactivity with a nonnatural molecule. This study illustrates the wealth of intrinsic survival mechanisms that can be exploited by bacteria when they are challenged with toxins.

• Alvarez-Buylla ER et al.
• Flower development.
• Arabidopsis Book. 2010; 8: 127-127
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• Flowers are the most complex structures of plants. Studies of Arabidopsis thaliana, which has typical eudicot flowers, have been fundamental in advancing the structural and molecular understanding of flower development. The main processes and stages of Arabidopsis flower development are summarized to provide a framework in which to interpret the detailed molecular genetic studies of genes assigned functions during flower development and is extended to recent genomics studies uncovering the key regulatory modules involved. Computational models have been used to study the concerted action and dynamics of the gene regulatory module that underlies patterning of the Arabidopsis inflorescence meristem and specification of the primordial cell types during early stages of flower development. This includes the gene combinations that specify sepal, petal, stamen and carpel identity, and genes that interact with them. As a dynamic gene regulatory network this module has been shown to converge to stable multigenic profiles that depend upon the overall network topology and are thus robust, which can explain the canalization of flower organ determination and the overall conservation of the basic flower plan among eudicots. Comparative and evolutionary approaches derived from Arabidopsis studies pave the way to studying the molecular basis of diverse floral morphologies.

• Doyle MR, Amasino RM
• A single amino acid change in the enhancer of zeste ortholog CURLY LEAF results in vernalization-independent, rapid flowering in Arabidopsis.
• Plant Physiol. 2009; 151: 1688-97
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• Many strains of Arabidopsis (Arabidopsis thaliana) require exposure to prolonged cold for rapid flowering, a process known as vernalization. Vernalization in Arabidopsis results in the suppression of FLOWERING LOCUS C (FLC), a repressor of flowering. In a screen for mutants that no longer require vernalization for rapid flowering, we identified a dominant allele of the Enhancer of Zeste E(z) ortholog CURLY LEAF (CLF), clf-59. CLF is a Polycomb Group gene, and the clf-59 mutant protein contains a proline-to-serine transition in a cysteine-rich region that precedes the SET domain. Mutant plants are early flowering and have reduced FLC expression, but, unlike clf loss-of-function mutants, clf-59 mutants do not display additional pleiotropic phenotypes. clf-59 mutants have elevated levels of trimethylation on lysine 27 of histone H3 (H3K27me3) at FLC. Thus, clf-59 appears to be a gain-of-function allele, and this allele represses FLC without some of the components required for vernalization-mediated repression. In the course of this work, we also identified a marked difference in H3K27me3 levels at FLC between plants that contain and those that lack the FRIGIDA (FRI) gene. Furthermore, FRI appears to affect CLF occupancy at FLC; thus, our work provides insight into the molecular role that FRI plays in delaying the onset of flowering.

• Wang Z, Yuan T, Yuan C, Niu Y, Sun D, Cui S
• LFR, which encodes a novel nuclear-localized Armadillo-repeat protein, affects multiple developmental processes in the aerial organs in Arabidopsis.
• Plant Mol Biol. 2009; 69: 121-31
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• The Armadillo (ARM)-repeat domain is a 42-amino acid protein-protein interaction motif present in many eukaryotic proteins. ARM-repeat proteins function in many cellular processes, including cytoskeletal regulation, nucleo-cytoplasmic trafficking, and transcriptional regulation. More than 100 genes encoding ARM-repeat proteins are predicted to exist in the Arabidopsis thaliana genome; however, most of them have unknown biological functions. Using map-based cloning, we isolated a novel recessive loss-of-function mutant, lfr-1, with developmental and morphological defects at the vegetative stage in the cotyledons and true leaves, and during the reproductive phase in the flowers and siliques. Complementation experiments and an analysis of the T-DNA insertion mutant lfr-2 revealed that LFR was responsible for all of the mutant phenotypes. LFR encodes a protein with three putative ARM-repeat domains that tends to cluster in the nucleus as discrete rounded speckles. LFR was broadly expressed while LFR was largely concentrated in the stem apex and root tip. Our data suggest that LFR is a novel nuclear-localized ARM-repeat protein that functions in leaf and flower development in Arabidopsis.

• Luque-Almagro VM et al.
• Characterization of the Pseudomonas pseudoalcaligenes CECT5344 Cyanase, an enzyme that is not essential for cyanide assimilation.
• Appl Environ Microbiol. 2008; 74: 6280-8
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• Cyanase catalyzes the decomposition of cyanate into CO(2) and ammonium, with carbamate as an unstable intermediate. The cyanase of Pseudomonas pseudoalcaligenes CECT5344 was negatively regulated by ammonium and positively regulated by cyanate, cyanide, and some cyanometallic complexes. Cyanase activity was not detected in cell extracts from cells grown with ammonium, even in the presence of cyanate. Nevertheless, a low level of cyanase activity was detected in nitrogen-starved cells. The cyn gene cluster of P. pseudoalcaligenes CECT5344 was cloned and analyzed. The cynA, cynB, and cynD genes encode an ABC-type transporter, the cynS gene codes for the cyanase, and the cynF gene encodes a novel sigma(54)-dependent transcriptional regulator which is not present in other bacterial cyn gene clusters. The CynS protein was expressed in Escherichia coli and purified by following a simple and rapid protocol. The P. pseudoalcaligenes cyanase showed an optimal pH of 8.5 degrees C and a temperature of 65 degrees C. An insertion mutation was generated in the cynS gene. The resulting mutant was unable to use cyanate as the sole nitrogen source but showed the same resistance to cyanate as the wild-type strain. These results, in conjunction with the induction pattern of the enzymatic activity, suggest that the enzyme has an assimilatory function. Although the induction of cyanase activity in cyanide-degrading cells suggests that some cyanate may be generated from cyanide, the cynS mutant was not affected in its ability to degrade cyanide, which unambiguously indicates that cyanate is not a central metabolite in cyanide assimilation.

• Xiang Y, Zou SC, Ou XL, Zhou MQ
• [Cloning of Rcet3, a novel gene related to family 2 cystatins].
• Nan Fang Yi Ke Da Xue Xue Bao. 2008; 28: 1151-3
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• OBJECTIVE: To clone the full-length Rcet3 gene, a novel gene related to family 2 cystatins, from mouse testis or other tissues. METHODS: Rcet3 gene was cloned using digital differential display (DDD) and RT-PCR was performed for cloning the full-length Rcet3 gene from adult mouse testis cDNA library with sequence analysis. RESULTS: Rcet3 cDNA was 610 bp in length, consisting of 4 exons to encode a protein with 140 amino acid residues. The encoded protein contained a potential signal peptide and a cystatin domain, but lacked critical consensus site important for cysteine protease inhibition. These characteristics could be seen in the Cres subgroup related to the family 2 cystatins. Rcet3 was specifically expressed in adult mouse testis, epididymis and the cerebrum, but at higher levels in the testis than in the epididymis and cerebrum. CONCLUSION: Rcet3 may be a new member of Cres subgroup of family 2 cystatins.

• Andersen SU et al.
• The conserved cysteine-rich domain of a tesmin/TSO1-like protein binds zinc in vitro and TSO1 is required for both male and female fertility in Arabidopsis thaliana.
• J Exp Bot. 2007; 58: 3657-70
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• Development of reproductive tissue and control of cell division are common challenges to all sexually reproducing eukaryotes. The Arabidopsis thaliana TSO1 gene is involved in both these processes. Mild tso1 mutant alleles influence only ovule development, whereas strong alleles have an effect on all floral tissues and cause cell division defects. The tso1 mutants described so far carry point mutations in a conserved cysteine-rich domain, the CRC domain, but the reason for the range of phenotypes observed is poorly understood. In the present study, the tesmin/TSO1-like CXC (TCX) proteins are characterized at the biochemical, genomic, transcriptomic, and functional level to address this question. It is shown that the CRC domain binds zinc, offering an explanation for the severity of tso1 alleles where cysteine residues are affected. In addition, the phylogenetic and expression analysis of the TCX genes suggested an overlap in function between AtTSO1 and the related gene AtTCX2. Their expression ratios indicated that pollen, in addition to ovules, would be sensitive to loss of TSO1 function. This was confirmed by analysis of novel tso1 T-DNA insertion alleles where the development of both pollen and ovules was affected.

• He Z, Jiang J, Hofmann MC, Dym M
• Gfra1 silencing in mouse spermatogonial stem cells results in their differentiation via the inactivation of RET tyrosine kinase.
• Biol Reprod. 2007; 77: 723-33
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• Spermatogenesis is the process by which spermatogonial stem cells divide and differentiate into sperm. The role of growth factor receptors in regulating self-renewal and differentiation of spermatogonial stem cells remains largely unclear. This study was designed to examine Gfra1 receptor expression in immature and adult mouse testes and determine the effects of Gfra1 knockdown on the proliferation and differentiation of type A spermatogonia. We demonstrated that GFRA1 was expressed in a subpopulation of spermatogonia in immature and adult mice. Neither Gfra1 mRNA nor GFRA1 protein was detected in pachytene spermatocytes and round spermatids. GFRA1 and POU5F1 (also known as OCT4), a marker for spermatogonial stem cells, were co-expressed in a subpopulation of type A spermatogonia from 6-day-old mice. In addition, the spermatogonia expressing GFRA1 exhibited a potential for proliferation and the ability to form colonies in culture, which is a characteristic of stem cells. RNA interference assays showed that Gfra1 small interfering RNAs (siRNAs) knocked down the expression of Gfra1 mRNA and GFRA1 protein in type A spermatogonia. Notably, the reduction of Gfra1 expression by Gfra1 siRNAs induced a phenotypic differentiation, as evidenced by the elevated expression of KIT, as well as the decreased expression of POU5F1 and proliferating cell nuclear antigen (PCNA). Furthermore, Gfra1 silencing resulted in a decrease in RET phosphorylation. Taken together, these data indicate that Gfra1 is expressed dominantly in mouse spermatogonial stem cells and that Gfra1 knockdown leads to their differentiation via the inactivation of RET tyrosine kinase, suggesting an essential role for Gfra1 in spermatogonial stem cell regulation.

• Ma Q et al.
• Molecular cloning and characterization of SRG-L, a novel mouse gene developmentally expressed in spermatogenic cells.
• Mol Reprod Dev. 2006; 73: 1075-83
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• Full-length cDNA of a novel mouse gene upregulated in late stages of spermatogenic cells was cloned from mouse testis using overlapping RT-PCR and RACE. The mRNA of the gene was expressed mainly in diplotene/pachytene spermatocytes, round and elongating spermatids. We named this gene as SRG-L (Spermatogenesis Related Gene expressed in late stages of spermatogenic cells, GenBank Accession No. AY352586). The tissue-specific analysis showed a higher expression level in testis and spleen. The gene is mapped on chromosome 8q33.1 and contains 18 exons. The full-length of cDNA is 2,843 bp with an open reading frame (ORF) of 2,625 bp that encodes a 104 kDa protein (874 amino acids) with a putative transmembrane region. The bioinformatics analysis revealed that the SRG-L has two conserved regions, transglutaminase-like homologues domain and D-serine dehydratase domain, rich phosphorylation sites and methylation sites. The SRG-L protein was detected in diplotene/pachytene spermatocytes and spermatids by immunohistochemical staining and Western blot. The results suggest that SRG-L may play definite roles regulating differentiation of germ cells during spermatogenesis, particularly during meiosis and spermiogenesis.

• Nie DS et al.
• Identification of a novel testis-specific gene mtLR1, which is expressed at specific stages of mouse spermatogenesis.
• Biochem Biophys Res Commun. 2005; 328: 1010-8
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• A novel testis-specific gene termed mtLR1 was identified by digital differential display. Sequence analyses revealed that mtLR1 protein contains an amino terminus leucine-rich repeat domain and shows 33% similarities to PIDD which functions in p53-mediated apoptosis. Northern blot analysis showed that mtLR1 mRNA was specifically expressed in adult mouse testis, and RT-PCR results also showed that mtLR1 was exclusively expressed in adult testis and not in spermatogonial cells. The expression of mtLR1 mRNA was developmentally upregulated in the testes during sexual maturation and was, conversely, downregulated by experimental cryptorchidism in vivo. We also showed that the expression of mtLR1 mRNA was relatively highly sensitive to heat stress in vitro. The green fluorescent protein produced by pEGFP-C3/mtLR1 was only detected in the cytoplasm of spermatogonia cell line GC-1 after 24 h posttransfection. Immunohistochemical analysis revealed that the protein is most abundant in the cytoplasm of spermatocytes and round spermatids within seminiferous tubules of the adult testis. The time-dependent expression pattern of mtLR1 in postnatal mouse testes suggested that mtLR1 gene might be involved in the regulation of spermatogenesis and sperm maturation.

• Jarvis S, Elliott DJ, Morgan D, Winston R, Readhead C
• Molecular markers for the assessment of postnatal male germ cell development in the mouse.
• Hum Reprod. 2005; 20: 108-16
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• BACKGROUND: A proliferation marker, proliferating cell nuclear antigen (PCNA), a Sertoli cell specific transcription factor, GATA-1 and the male germ cell specific, RNA binding motif (RBM), were used to identify different cellular populations during postnatal development of the mouse testis. METHODS: Immunohistochemistry, RT-PCR and real-time quantitative RT-PCR (QRT-PCR) were used. RESULTS: PCNA was expressed in pre-Sertoli and germ cells on the day of birth. Both pre-meiotic germ cells and spermatocytes expressed RBM throughout postnatal development. RBM-positive cell counts and QRT-PCR of RBM showed that average level of RBM per cell is highest in juvenile males between 14 and 21 days. From 42 days onward, there was a dramatic decrease in RBM expression in individual pre-meiotic and meiotic germ cells. CONCLUSIONS: These markers were used to correlate cell proliferative capability, gene expression profile and anatomic location within the developing mouse testis. The majority of germ cells start active proliferation once they have migrated to the basement membrane or immediately before. RBM is more highly expressed during the first wave of spermatogenesis versus subsequent waves, suggesting that there may be a change in the activity of RBM.

• Gomes C et al.
• Expression of the putative sterol binding protein Stard6 gene is male germ cell specific.
• Biol Reprod. 2005; 72: 651-8
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• Mammalian spermatogenesis is orchestrated by many specific molecular and cellular events. To understand the detailed mechanism by which spermatogenesis is controlled, the specific genes involved in this process must be identified and studied. From the subtracted cDNA library of rat testis prepared using the representational difference analysis (RDA) method, we isolated the cDNA clone of steroidogenic acute regulatory (StAR) protein-related lipid transfer (START) protein 6 (Stard6). Stard6 cDNA consists of 1146 base pairs of nucleotides and has the longest open reading frame, of 227 amino acids. Northern blot analysis revealed Stard6 mRNA to be testis-specific. The mRNA transcript appeared from the third week of postnatal development, and the expression level increased up to adulthood. Moreover, in situ hybridization showed Stard6 mRNA expression to be germ cell-specific and expressed only during the maturation stages of round and elongated spermatids of adult rat testis. Western blot analysis with Stard6 antibody revealed a 28-kDa Stard6 protein only in testis. Immunohistochemistry further confirmed localization of Stard6 protein expressed in mature germ cells, in concert with the in situ hybridization result. Taken together, these results suggest that Stard6, a member of the START protein family, may play a role during germ cell maturation in adult rat testis.

• Nie DS et al.
• Molecular cloning and primary functional analysis of a novel human testis-specific gene.
• Yi Chuan Xue Bao. 2005; 32: 337-45
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• In this study, a new data mining tool called Digital Differential Display (DDD) from the NCBI was used to predict testis-specific expressed genes from the expressed sequence tag (EST) database. DDD (digital differential display) was performed between nine testis libraries and seventy libraries derived from other tissues. We identified a new contig of ESTs (HS. 326528) which was from testis libraries. To validate the use of bioinformatic approaches in gene discovery, the ESTs (HS. 326528), which were predicted to be testis-specific, were chosen for further study. Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) analysis of matched sets of cDNAs from testis and other tissues indicated that the ESTs were specifically expressed in testis. This result was further validated by multi-tissue Northern blot. The full-length cDNA encompassing the entire open reading frame was cloned and, in view of its apparent specificity to testis, the gene was termed homo sapiens spermatogenesis-related gene 8---SRG8 (GenBank accession number: AY489187). The gene whose full cDNA length is 1 044 bp containing 3 exons and 2 introns is located in human chromosome 15q26.2, the cDNA encodes a novel protein of 105 amino acides with a theoretical molecular weight of 11.7 kD and isoelectric point of 10.09 which shares no significant homology with any known proteins in database. Real time PCR analysis of testis of different developmental periods revealed that SRG8 gene is significantly expressed in adult testis. The green fluorescent protein produced by pEGFP-C3/SRG8 was detected in the nucleus of HeLa cells after 24 h post-transfection. Cell cycle analysis showed that SRG8 can accelerate HeLa cells to traverse the S-phase and enter the G2-phase compared with the control without transfection of SRG8, which suggested that this gene plays an important role in the development of testis. The discovery of SRG8 showed that DDD combined with experiments is a feasible, time-saving strategy to identify new candidate genes for testis-specific development

• Zhou Y, Zhao Q, Bishop CE, Huang P, Lu B
• Identification and characterization of a novel testicular germ cell-specific gene Ggnbp1.
• Mol Reprod Dev. 2005; 70: 301-7
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• A novel gene Ggnbp1 was identified during yeast two-hybrid screening of gametogenetin protein 1 (GGN1)-interacting proteins. Ggnbp1 gene was found in mouse, rat, and human genomes but not in sequenced yeast, worms, fly, or fish genomes. Northern blotting analysis revealed that the gene was specifically expressed in the testis but not expressed in the other tissues. In situ hybridization showed that it was testicular germ cell-specific and was specifically expressed in later primary spermatocytes, meiotic cells, and early round spermatids. Western blotting analysis detected a protein of expected size in and only in the testis. By making membrane and cytosolic fractions of germ cells, we were able to show that GGNBP1 associated with the membrane. The identification and characterization of a novel germ cell-specific gene Ggnbp1 is the first step toward the defining of the functions of Ggnbp1 in spermatogenesis.

• Narita NN et al.
• Overexpression of a novel small peptide ROTUNDIFOLIA4 decreases cell proliferation and alters leaf shape in Arabidopsis thaliana.
• Plant J. 2004; 38: 699-713
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• Leaf shape is determined by polar cell expansion and polar cell proliferation along the leaf axes. However, the genes controlling polar cell proliferation during leaf morphogenesis are largely unknown. We identified a dominant mutant of Arabidopsis thaliana, rotundifolia4-1D (rot4-1D), which possessed short leaves and floral organs. We showed that the altered leaf shape is caused by reduced cell proliferation, specifically in the longitudinal (proximal-distal) axis of the leaf, suggesting that the ROT4 gene controls polar cell proliferation in lateral organs. The ROT4 open-reading frame (ORF) encodes a novel small peptide that had not been identified in the Arabidopsis genome annotation. Overexpression of a ROT4-green fluorescence protein (GFP) fusion protein in transgenic plants recapitulated the rot4 phenotype, suggesting that ROT4 acts to restrict cell proliferation. The ROT4-GFP fusion protein localized to the plasma membrane when expressed in transgenic Arabidopsis plants. Phylogenetic analysis indicates that ROT4 defines a novel seed plant-specific family of small peptides with 22 members in Arabidopsis, ROT FOUR LIKE1-22 (RTFL1-22). All RTFL members share a conserved 29-amino acid domain, the RTF domain, and overexpression of the ROT4 RTF domain alone is sufficient to confer a rot4-1D phenotype. Loss-of-function mutations in several RTFL genes were aphenotypic, suggesting that there may be some functional redundancy between family members. Analyses by reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization revealed that ROT4 is expressed in the shoot apex and young leaves of wild-type plants, consistent with a role for ROT4 in controlling polarity-dependent cell proliferation during wild-type leaf morphogenesis.

• Guo R et al.
• Stage-specific and tissue-specific expression characteristics of differentially expressed genes during mouse spermatogenesis.
• Mol Reprod Dev. 2004; 67: 264-72
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• Spermatogenesis occurs in successive mitotic, meiotic, and post-meiotic phase, and involves a number of unique processes including meiosis and dramatic morphological changes. The unique differentiation mechanisms of spermatogenesis suggest the existence of germ-cell-specific molecules. The most straight forward strategy to elucidate differentiation mechanisms is to identify and characterize differentiation-specific molecules and their associated genes in germ cells. However, only a few genes specifically involved in spermatogenesis have been studied. In the present study, six different types of spermatogenic cells (primitive type A spermatogonia, type B spermatogonia, preleptotene spermatocytes, pachytene spermatocytes, round spermatids, and elongating spermatids) were isolated from Balb/c mice testes using velocity sedimentation and Atlas cDNA arrays containing 1,176 known mouse genes were used to determine the gene expression profiles of the spermatogenic cells. The expression of 260 genes were detected in six different stages of spermatogenic cells and a number of genes showed differential expression. The 23 differentially expressed genes were further analysed by reverse transcription polymerase chain reaction (RT-PCR) for their stage-specific and tissue-specific expression characteristics. Based on the results of RT-PCR, six genes highly express in both primitive type A and type B spermatogonia, four genes up-regulate in type B spermatogonia, two genes up-regulate in spermatocytes, two genes up-regulate in spermatids, three genes express constantly from primitive A spermatogonia to elongating spermatids, two genes express constantly from primitive A spermatogonia to round spermatids, two genes do not change in their expression during spermatogenesis, two genes can be detected highly in adult testis, but are undetectable in spermatogenic cells. The tissue-specific expression characteristics of the 23 genes showed that some of them specifically expressed in testes or other tissues. These data provide new information for further studies into spermatogenesis-related genes and may lead to the identification of genes with potential relevance to the differentiation of spermatogenic cells. In addition, some of these genes could be considered to be used as the molecular markers for different stages of spermatogenic cells.

• Buschmann H et al.
• Helical growth of the Arabidopsis mutant tortifolia1 reveals a plant-specific microtubule-associated protein.
• Curr Biol. 2004; 14: 1515-21
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• Plants can grow straight or in the twisted fashion exhibited by the helical growth of some climbing plants. Analysis of helical-growth mutants from Arabidopsis has indicated that microtubules are involved in the expression of the helical phenotype. Arabidopsis mutants growing with a right-handed twist have been reported to have cortical microtubules that wind around the cell in left-handed helices and vice versa. Microtubular involvement is further suspected from the finding that some helical mutants are caused by single amino acid substitutions in alpha-tubulin and because of the sensitivity of the growth pattern to anti-microtubule drugs. Insight into the roles of microtubules in organ elongation is anticipated from analyses of genes defined by helical mutations. We investigated the helical growth of the Arabidopsis mutant tortifolia1/spiral2 (tor1/spr2), which twists in a right-handed manner, and found that this correlates with a complex reorientation of cortical microtubules. TOR1 was identified by a map-based approach; analysis of the TOR1 protein showed that it is a member of a novel family of plant-specific proteins containing N-terminal HEAT repeats. Recombinant TOR1 colocalizes with cortical microtubules in planta and binds directly to microtubules in vitro. This shows that TOR1 is a novel, plant-specific microtubule-associated protein (MAP) that regulates the orientation of cortical microtubules and the direction of organ growth.

• Arango NA, Pearson EJ, Donahoe PK, Teixeira J
• Genomic structure and expression analysis of the mouse testis-specific ribbon protein (Trib) gene.
• Gene. 2004; 343: 221-7
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• During our analyses of genes required for the development and function of the mouse gonads, we identified a novel testis-specific mRNA, transcribed from a gene that we have named testis-specific ribbon protein (Trib). In the mouse, Trib is located on chromosome 15, overlapping with and transcribed in the opposite orientation of the meiosis specific gene Smc1beta. The deduced amino acid sequence of testis ribbon (TRIB) protein is highly conserved between human, mouse, and rat and contains the ribbon motifs found in the largely uncharacterized microtubule ribbon protein ribbon43a (RIB43A). We show by Northern blot analyses and reverse transcription-polymerase chain reaction (RT-PCR) that Trib mRNA is specifically expressed in the adult testis. In situ hybridization indicates that Trib is expressed solely in germ cells during the leptotene-pachytene stages of spermatogenesis. The high level of evolutionary conservation and the cellular and temporal expression suggest that Trib may be required for mouse spermatogenesis and male fertility. Here, we describe the genomic structure and expression profile of mouse Trib and compare its homology with other ribbon proteins.

• Xiang Y, Nie DS, Lu GX
• Cloning of a novel gene, Cymg1, related to family 2 cystatins and expressed at specific stages of mouse testis development.
• J Genet. 2004; 83: 257-63
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• We have cloned a novel gene, Cymg1 (GenBank accession number AY600990), from a mouse testis cDNA library. Cymg1 is located in 2G3 of mouse chromosome 2. The cDNA includes an open reading frame that encodes 141 amino acid residues. The encoded polypeptide has a cysteine protease inhibitor domain found in the family 2 cystatins but lacks critical consensus sites important for cysteine protease inhibition. These characteristics are seen in the proteins of the CRES subfamily of the family 2 cystatins which are expressed specifically in the reproductive tract. CYMG1 protein shows 44% identity with mouse CRES and 30% identity with mouse cystatin C. Northern blot analysis showed that the Cymg1 gene was specifically expressed in adult mouse testis. RT-PCR also showed that Cymg1 was expressed in testis and spermatogonial cells. Cymg1 expression level varied in the different developmental stages of mouse testis, and were coincidental with spermatogenesis and sex maturation. These results indicate that Cymg1 may play important roles in mouse spermatogenesis and sex maturation.

• Adams-Phillips L, Barry C, Kannan P, Leclercq J, Bouzayen M, Giovannoni J
• Evidence that CTR1-mediated ethylene signal transduction in tomato is encoded by a multigene family whose members display distinct regulatory features.
• Plant Mol Biol. 2004; 54: 387-404
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• Ethylene governs a range of developmental and response processes in plants. In Arabidopsis thaliana, the Raf-like kinase CTR1 acts as a key negative regulator of ethylene responses. While only one gene with CTR1 function apparently exists in Arabidopsis, we have isolated a family of CTR1- like genes in tomato ( Lycopersicon esculentum ). Based on amino acid alignments and phylogenetic analysis, these tomato CTR1- like genes are more similar to Arabidopsis CTR1 than any other sequences in the Arabidopsis genome. Structural analysis reveals considerable conservation in the size and position of the exons between Arabidopsis and tomato CTR1 genomic sequences. Complementation of the Arabidopsis ctr1-8 mutant with each of the tomato CTR genes indicates that they are all capable of functioning as negative regulators of the ethylene pathway. We previously reported that LeCTR1 expression is up-regulated in response to ethylene. Here, quantitative real-time PCR was carried out to detail expression for LeCTR1 and the additional CTR1 -like genes of tomato. Our results indicate that the tomato CTR1 gene family is differentially regulated at the mRNA level by ethylene and during stages of development marked by increased ethylene biosynthesis, including fruit ripening. The possibility of a multi-gene family of CTR1 -like genes in other species besides tomato was examined through mining of EST and genomic sequence databases.

• Gimenez R, Nunez MF, Badia J, Aguilar J, Baldoma L
• The gene yjcG, cotranscribed with the gene acs, encodes an acetate permease in Escherichia coli.
• J Bacteriol. 2003; 185: 6448-55
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• We isolated an Escherichia coli mutant strain that suppresses the glycolate-negative phenotype of a strain deficient in both GlcA and LldP transporters of this compound. This suppressing phenotype was assigned to yjcG, a gene whose function was previously unknown, which was found to encode a membrane protein able to transport glycolate. On the basis of sequence similarity, the yjcG gene product was classified as a member of the sodium:solute symporter family. Northern experiments revealed that yjcG is cotranscribed with its neighbor, acs, encoding acetyl coenzyme A synthetase, which is involved in the scavenging acetate. The fortuitous presence of an IS2 element in acs, which impaired yjcG expression by polarity in our parental strain, allowed us to conclude that the alternative glycolate carrier became active after precise excision of IS2 in the suppressed strain. The finding that yjcG encodes a putative membrane carrier for glycolate and the cotranscription of yjcG with acs suggested that the primary function of the yjcG gene product (proposed gene name, actP) could be acetate transport and allowed us to define an operon involved in acetate metabolism. The time course of [1,2-(14)C]acetate uptake and the results of a concentration kinetics analysis performed with cells expressing ActP or cells deficient in ActP supported the the hypothesis that this carrier is an acetate transporter and suggested that there may be another transport system for this monocarboxylate.

• Hwang KC, Ok DW, Hong JC, Kim MO, Kim JH
• Cloning, sequencing, and characterization of the murine nm23-M5 gene during mouse spermatogenesis and spermiogenesis.
• Biochem Biophys Res Commun. 2003; 306: 198-207
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• Nucleoside diphosphate kinases (NDPKs) are conserved throughout evolution and have been shown to be involved in various biological phenomena. By functional screening in yeast, we identified a new member of the NDPK family, nm23-M5, which encodes a 211-amino acid protein with 86% identity to the human homolog Nm23-H5. Northern blot analysis revealed that nm23-M5 encodes two transcripts of 0.8 and 0.7kb, which are highly and specifically expressed in adult testis. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis showed that nm23-M5 transcripts first appear in pachytene spermatocytes and increase in abundance in subsequent stages. However, a low level of nm23-M5 mRNA was detected by RT-PCR in other tissues, such as ovary, brain, heart, and kidney. In situ hybridization studies showed that testicular nm23-M5 transcripts are localized in stage 12 to stage 16 spermatids in the neighboring lumen of seminiferous tubules. This distribution contrasts with that of Nm23-H5 transcripts, which are specifically found in spermatogonia and early spermatocytes. The heterologous expression of nm23-M5 in yeast cells confers protection from cell death induced by Bax, which is due to the generation of reactive oxygen species. Furthermore, overexpression of nm23-M5 in fibroblasts altered the cellular levels of several antioxidant enzymes, particularly glutathione peroxidase 5. Thus, we believe that the murine nm23-M5 gene plays an important role in late spermiogenesis by elevating the ability of late-stage spermatids to eliminate reactive oxygen species.

• Sorensen AM, Krober S, Unte US, Huijser P, Dekker K, Saedler H
• The Arabidopsis ABORTED MICROSPORES (AMS) gene encodes a MYC class transcription factor.
• Plant J. 2003; 33: 413-23
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• Visual screening of a T-DNA mutagenised population of Arabidopsis thaliana for an absence of silique elongation lead to the isolation of the aborted microspores (ams) mutant that shows a sporophytic recessive male sterile phenotype. Homozygous mutant plants are completely devoid of mature pollen. Pollen degeneration occurs shortly after release of the microspores from the tetrad, prior to pollen mitosis I. Premature tapetum and microspore degeneration are the primary defects caused by this lesion, while a secondary effect is visualised in the stamen filaments, which are reduced in length and lie beneath the receptive stigma at flower opening. The disrupted gene was isolated and revealed a T-DNA element to be inserted into the eighth exon of a basic helix-loop-helix (bHLH) gene located on chromosome II. This protein sequence contains a basic DNA binding domain and two alpha helices separated by a loop, typical of a transcription factor belonging to the MYC sub family of bHLH genes. Therefore, AMS plays a crucial role in tapetal cell development and the post-meiotic transcriptional regulation of microspore development within the developing anther.

• Noh SJ, Kwon CS, Oh DH, Moon JS, Chung WI
• Expression of an evolutionarily distinct novel BiP gene during the unfolded protein response in Arabidopsis thaliana.
• Gene. 2003; 311: 81-91
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• Compared to mammals, little is known about the unfolded protein response (UPR) in plants. Using an oligonucleotide array comprising approximately 8200 Arabidopsis genes we investigated the effect of endoplasmic reticulum (ER) stress on gene expression. Expression of 26 genes increased, including at least nine whose products act in the ER, while their transcriptional activations were confirmed by promoter analyses. Among them, BiP-L, a novel BiP, whose expression appeared to be regulated by two promoter sequences perfectly matching mammalian ERSE. Cloning and sequencing of full-length BiP-L cDNA showed it contained a signal peptide sequence and the ER retention signal (HDEL). Interestingly, BiP-L was substantially different from the other two Arabidopsis BiP genes in genomic organization and sequence homology. Furthermore, phylogenetic analysis showed that the BiP-L protein is the most distal form among the reported plant BiP proteins. RNA levels of BiP-L were very low in various mature Arabidopsis plant organs, while significant levels of BiP-L only observed in stressed seedlings. Transcription of BiP-L during ER stress was shown to be regulated by a feedback loop.

• Thakur JK et al.
• A POLYCOMB group gene of rice (Oryza sativa L. subspecies indica), OsiEZ1, codes for a nuclear-localized protein expressed preferentially in young seedlings and during reproductive development.
• Gene. 2003; 314: 1-13
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• The SET domains are conserved amino acid sequences present in chromosomal proteins that contribute to the epigenetic control of gene expression by altering regional organization of the chromatin structure. The SET domain proteins are divided into four subgroups as categorized by their Drosophila members; enhancer of zeste (E(Z)), trithorax (TRX), absent small or homeotic 1 (ASH1) and supressor of variegation (SU(VAR)3-9). Homologs of all four classes have been characterized in yeast, mammals and plants. We report here the isolation and characterization of rice (Oryza sativa L. subspecies indica) cDNA, OsiEZ1, as a monocot member of this family. The OsiEZ1 cDNA is 3133 bp long with an ORF of 2799 bp, and the predicted amino acid sequence (895 residues) corresponds to a protein of ca. 98 kDa. All the characteristic domains known to be conserved in E(Z) homologs (subgroup I) of SET domain containing proteins are present in OsiEZ1. In the rice genome, a 7499 bp long OsiEZ1 sequence is split into 17 exons interrupted by 16 introns. Southern analysis indicates that OsiEZ1 is represented as single copy in the rice genome. Expression studies revealed that the OsiEZ1 transcript level was highest in rice flowers, almost undetectable in developing seeds of 1-2 days post-fertilization but increased significantly in young seeds of 3-5 days post-fertilization. The OsiEZ1 transcript was barely detectable in mature zygotic embryos, but its levels were significantly higher in callus derived from rice scutellum, somatic embryos and young seedlings. The OsiEZ1/GUS recombinant protein was confined to the nucleus in living cells of particle-bombarded onion peels. The expression of OsiEZ1 complemented a set1Delta Saccharomyces cerevisiae mutant that is impaired in telomeric silencing. We suggest that the nuclear-localized OsiEZ1 has a role in regulating various aspects of plant development, and this control is most likely brought about by repressing the activity of downstream regulatory genes.

• Sutou S, Miwa K, Matsuura T, Kawasaki Y, Ohinata Y, Mitsui Y
• Native tesmin is a 60-kilodalton protein that undergoes dynamic changes in its localization during spermatogenesis in mice.
• Biol Reprod. 2003; 68: 1861-9
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• Tesmin is a testis-specific protein. Four mouse tesmin cDNAs so far reported encode a testis-specific, metallothionein-like, 30-kDa protein (tesmin-30). An antibody against tesmin-30, however, detected a protein of 60 kDa (tesmin-60) from the mouse testis. To resolve the relationship between the two, the immunoprecipitated native tesmin-60 was sequenced. The result indicated that tesmin-30 is not full-length but is part of the C-terminal half of tesmin-60. The full-length cDNA (2.2 kilobases [kb]) encoding tesmin-60 (475 amino acid residues) and its genomic DNA (23 kb) were cloned and sequenced. A search of databases indicated that tesmin is a member of the CXC-hinge-CXC family. Immunohistochemistry indicated that tesmin exhibits dynamic subcellular localization changes during spermatogenesis. Before meiosis, it was localized in the cytoplasm of early to late spermatocytes and then translocated into the nucleus just before meiotic division. After meiosis, it appeared in spermatids, starting from the acrosomal vesicles, moving to the nuclear membrane and then to the caudal end as the spermatids elongated, and finally relocating into the cytoplasm. Oxidative stress by cobalt chloride, as well as by diethylmaleate, induced both premature translocation of tesmin from the cytoplasm to the nucleus and apoptotic signals in spermatocytes. The persistent existence of tesmin and its temporally and spatially dynamic localization suggest that tesmin is involved in multiple stages of spermatogenesis and spermiogenesis, possibly during sperm maturation and/or morphogenesis.

• Zielinski RE
• Characterization of three new members of the Arabidopsis thaliana calmodulin gene family: conserved and highly diverged members of the gene family functionally complement a yeast calmodulin null.
• Planta. 2002; 214: 446-55
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• Three genes encoding members of the EF-hand family of Ca2+-binding proteins were identified from Arabidopsis thaliana (L.) Heynh. sequences deposited in the expressed sequence tag and genomic sequence databases. Full-length cDNAs for each of the genes, Cam7, Cam8, and Cam9, were sequenced. Cam7 encodes a conventional 16.8-kDa, 148-amino-acid calmodulin protein (CaM). In contrast, Cam8 and 9 encode highly diverged isoforms of the protein that share 73 and 49% amino acid sequence identity, respectively, with CaM7. RNA gel blot and reverse transcription-polymerase chain reaction experiments revealed that each of the genes is expressed in leaves, flowers and siliques. To test the functional properties of the polypeptides encoded by these genes, they were expressed in Escherichia coli and the yeast Saccharomyces cerevisiae. Each was purified by Ca2+-dependent hydrophobic affinity chromatography. CaM7, but neither CaM8 nor CaM9, formed a complex with a basic amphiphilic helical peptide in the presence of Ca2+ that could be identified by gel electrophoresis. In spite of these in vitro differences, each of the sequences functionally substituted for yeast CMD1 to maintain viability. Isolation of yeast strains complemented by Cam9 required selection against the plasmid harboring wild-type yeast sequences, whereas complementation by Cam7 and Cam8 did not. These results suggest that the mechanism of action of CaM8 and CaM9 is similar to that of more conventional CaM sequences. CaM9, and to a lesser degree CaM8, however, appear to represent Ca2+-binding sensor proteins that interact with a more limited set of target proteins than do more conventional CaM isoforms.

• Twell D et al.
• MOR1/GEM1 has an essential role in the plant-specific cytokinetic phragmoplast.
• Nat Cell Biol. 2002; 4: 711-4
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• MOR1 is a member of the MAP215 family of microtubule-associated proteins and is required to establish interphase arrays of cortical microtubules in plant cells. Here we show that MOR1 binds microtubules in vivo, localizing to both cortical microtubules and to areas of overlapping microtubules in the phragmoplast. Genetic complementation of the cytokinesis-defective gemini pollen 1-1 (gem1-1) mutation with MOR1 shows that MOR1 (which is synonymous with the protein GEM1) is essential in cytokinesis. Phenotypic analysis of gem1-1 and gem1-2, which contains a T-DNA insertion, confirm that MOR1/GEM1 is essential for regular patterns of cytokinesis. Both the gem1-1 and gem1-2 mutations cause the truncation of the MOR1/GEM1 protein. In addition, the carboxy-terminal domain of the protein, which is absent in both mutants, binds microtubules in vitro. Our data show that MOR1/GEM1 has an essential role in the cytokinetic phragmoplast.

• Tanaka K et al.
• Spermatogonia-dependent expression of testicular genes in mice.
• Dev Biol. 2002; 246: 466-79
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• Spermatogenesis is initiated by the interaction of germ cells and somatic cells in seminiferous tubules. We used cDNA microarrays and representational difference analysis to identify genes that are expressed in the testis of the jsd/jsd mutant mouse, which contains only type A spermatogonial germ cells and Sertoli cells, but not in the testis of the W/W(v) mutant mouse, where Sertoli cells but few germ cells are present. We isolated 20 known genes and 4 novel genes, including 2 genes encoding lipocalin family members (prostaglandin D synthetase and 24p3) and 2 tumor suppressors (protein tyrosine phosphatase TD14 and Sui1). All 24 of these jsd/jsd-derived genes were highly expressed in the cryptorchid testis as well as in the jsd/jsd testis. This indicates that their selective expression is not directly caused by the as-yet-uncharacterized jsd gene product, but is rather correlated to the cessation of spermatogonial differentiation. In situ hybridization analysis and flow cytometric sorting followed by reverse transcriptase-PCR revealed that these genes are expressed in both the spermatogonial germ cells and the somatic cells in the developing gonads and adult testes. As the mRNAs of these jsd/jsd-derived genes were barely detectable in the W/W(v) testis, we propose that early spermatogonial germ cells regulate the expression of a group of testicular genes.

• Zhang F et al.
• Characterization of Glis2, a novel gene encoding a Gli-related, Kruppel-like transcription factor with transactivation and repressor functions. Roles in kidney development and neurogenesis.
• J Biol Chem. 2002; 277: 10139-49
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• In this study, we describe the characterization of a gene encoding a novel Kruppel-like protein, named Glis2. Glis2 encodes a relatively proline-rich, basic 55.8-kDa protein. Its five tandem Cys(2)-His(2) zinc finger motifs exhibit the highest homology to those of members of the Gli and Zic subfamilies of Kruppel-like proteins. Confocal microscopic analysis demonstrated that Glis2 localizes to the nucleus. Analysis of the genomic structure of the Glis2 gene showed that it is composed of 6 exons separated by 5 introns spanning a genomic region of more than 7.5 kb. Fluorescence in situ hybridization mapped the mouse Glis2 gene to chromosome 16A3-B1. Northern blot analysis showed that the Glis2 gene encodes a 3.8-kb transcript that is most abundant in adult mouse kidney. By in situ hybridization, expression was localized to somites and neural tube, and during metanephric development predominantly to the ureteric bud, precursor of the collecting duct, and inductor of nephronic tubule formation. One-hybrid analysis using Glis2 deletion mutants identified a novel activation function (AF) at the N terminus. The activation of transcription through this AF domain was totally suppressed by two repressor functions just downstream from the AF. One of the repressor functions is contained within the first zinc finger motif. The level of transcriptional activation and repression varied with the cell line tested, which might be due to differences in cell type-specific expression of co-activators and co-repressors. Our results suggest that Glis2 behaves as a bifunctional transcriptional regulator. Both the activation and repressor functions may play an important role in the regulation of gene expression during embryonic development.

• Yan W, Burns KH, Ma L, Matzuk MM
• Identification of Zfp393, a germ cell-specific gene encoding a novel zinc finger protein.
• Mech Dev. 2002; 118: 233-9
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• Using the digital differential display program of the National Center for Biotechnology Information, we identified a contig of expression sequence tags (ESTs) which were unique to ovary, testis, and egg libraries. The full-length cDNA of this transcript was deduced and further confirmed by reverse transcriptase polymerase chain reaction (RT-PCR). The cDNA encodes a novel protein of 341 amino acids with a nuclear localization signal. The carboxyl-terminus of the protein contains three C2H2 zinc fingers, and the NH(2)-terminus is proline and serine-rich. Based on the conserved zinc finger motifs, we have termed this novel protein as zinc finger protein 393 (ZFP393). Northern blot and RT-PCR analyses revealed that Zfp393 mRNA was exclusively expressed in testis and ovary. The expression sites were further localized by in situ hybridization to step 3-8 spermatids in testis and growing oocytes in ovary. The Zfp393 gene consists of three exons spanning approximately 8 kb on the distal part of mouse chromosome 4. The carboxyl-terminal zinc finger region is highly homologous to several zinc finger-containing proteins, but no proteins were found to share sequence similarity with the NH(2)-terminal region of ZFP393. Genomic database mining and Southern blot analysis indicate that Zfp393 is a single copy gene. We hypothesize that ZFP393 functions as a germ cell-specific transcription factor that plays important roles in spermatid differentiation and oocyte development.

• Fukaki H, Tameda S, Masuda H, Tasaka M
• Lateral root formation is blocked by a gain-of-function mutation in the SOLITARY-ROOT/IAA14 gene of Arabidopsis.
• Plant J. 2002; 29: 153-68
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• Lateral root development is a post-embryonic organogenesis event that gives rise to most of the underground parts of higher plants. Auxin promotes lateral root formation, but the molecular mechanisms involved are still unknown. We have isolated a novel Arabidopsis mutant, solitary-root (slr), which has reduced sensitivity to auxin. This dominant slr-1 mutant completely lacks lateral roots, and this phenotype cannot be rescued by the application of exogenous auxin. Analysis with cell-cycle and cell-differentiation markers revealed that the slr-1 mutation blocks cell divisions of pericycle cells in lateral root initiation. The slr-1 mutant is also defective in root hair formation and in the gravitropic responses of its roots and hypocotyls. Map-based positional cloning and isolation of an intragenic suppressor mutant revealed that SLR encodes IAA14, a member of the Aux/IAA protein family. Green fluorescent protein-tagged mutant IAA14 protein was localized in the nucleus, and the gain-of-function slr-1/iaa14 mutation decreased auxin-inducible BA-GUS gene expression in the root, suggesting that SLR/IAA14 acts as a transcriptional repressor. These observations indicate that SLR/IAA14 is a key regulator in auxin-regulated growth and development, particularly in lateral root formation.

• Li Y, Friel PJ, Robinson MO, McLean DJ, Griswold MD
• Identification and characterization of testis- and epididymis-specific genes: cystatin SC and cystatin TE-1.
• Biol Reprod. 2002; 67: 1872-80
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• Differential display-reverse transcriptase-polymerase chain reaction was used to examine Sertoli cell gene expression. As a result, two new members of the mouse cystatin multigene family were isolated and named cystatin SC (cystatin-related gene expressed in Sertoli cells) and cystatin TE-1 (cystatin-related gene highly expressed in testis and epididymis). The full-length cDNA sequence of cystatin SC contains an open reading frame that encodes a putative signal peptide of 20 amino acids and a mature protein of 110 amino acids, whereas that of cystatin TE-1 encodes a 128 amino acid protein with a predicted signal peptide of 21 amino acids. Both of the deduced amino acid sequences contain four highly conserved cysteine residues in precise alignment with other cystatin family members. The derived cystatin SC and TE-1 amino acid sequences lack some of the specific, highly conserved motifs believed to be necessary for cysteine proteinase inhibition activity. Northern blot analysis revealed that cystatin SC mRNA was detected only in the testis, whereas the cystatin TE-1 gene was highly expressed in testis and epididymis with very low expression in ovary and prostate. In situ hybridization showed that cystatin SC mRNA was localized mainly to Sertoli cells with an obvious stage-dependent expression, and that cystatin TE-1 mRNA was predominantly expressed in Sertoli cells without apparent stage-dependent expression. Cystatin TE-1 mRNA, as displayed by in situ hybridization, was expressed only in the epithelial cells of the proximal caput region of the epididymis. The unusual amino acid sequence and highly restricted expression suggests that cystatins SC and TE-1 play a very specialized role in the testis and epididymis.

• Meccariello R et al.
• Mouse sperm cell-specific DnaJ first homologue: an evolutionarily conserved protein for spermiogenesis.
• Biol Reprod. 2002; 66: 1328-35
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• Msj-1 (mouse sperm cell-specific DnaJ first homologue) is a gene specifically expressed in germ cells at haploid stages. The protein first appears in round spermatids, accumulates in the periacrosomal region of elongating spermatids, and is maintained in spermatozoa. The msj-1 expression pattern is consistent with a role for this DnaJ protein in the spermiogenesis process. In this study, we used two experimental models, the anuran amphibian Rana esculenta and the wobbler mutant mouse, to explore the role of MSJ-1 during spermatogenesis, with a focus on spermiogenesis. Mice homozygous for the recessive mutation wobbler (wr/wr), a mutation of unknown identity, produce sperm cells characterized by a missing acrosome. In Rana esculenta testis, detection of high levels of MSJ-1 protein coincided with the appearance of postmeiotic germ cells during the annual sexual cycle. Conversely, elimination of the meiotic and postmeiotic stages, through gonadotropin administration at low temperature, abolished the MSJ-1 immunoreactive signal. In 20-day-old mice, when postmeiotic germ cells appeared for the first time, MSJ-1 mRNA and protein were observed in +/+ testis but were barely detectable in wr/wr testis. In adult testis, reduced MSJ-1 protein levels were observed in both +/wr and wr/wr testis, as compared with +/+ controls. Similarly, numbers of spermatids that stained by immunofluorescence for MSJ-1 appeared to be progressively reduced in adult +/+, +/wr, and wr/wr mouse testes, respectively. Characterization of the endocrine status of wobbler testis revealed reduced transcript levels of estrogen receptor alpha and reduced intratesticular androgen levels. However, androgen treatment did not affect MSJ-1 protein levels in either frogs or mice. In conclusion, our data in Rana esculenta and the wobbler mouse demonstrate a tight correlation between MSJ-1 protein expression and postmeiotic stages. In particular, the findings in the wobbler testis suggest a role for this protein in acrosomogenesis.

• Muller S, Fuchs E, Ovecka M, Wysocka-Diller J, Benfey PN, Hauser MT
• Two new loci, PLEIADE and HYADE, implicate organ-specific regulation of cytokinesis in Arabidopsis.
• Plant Physiol. 2002; 130: 312-24
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• In screens for regulators of root morphogenesis in Arabidopsis we isolated six new recessive mutants with irregular cell expansion. Complementation analyses placed the mutations in two loci, PLEIADE (PLE) and HYADE (HYA). Phenotypic analyses revealed multinucleated cells, cell wall stubs, and synchronized cell divisions in incompletely separated cells that are all characteristics of defective cytokinesis. These defects were pronounced in roots and undetectable in aerial organs. In addition, fertility and germination were not affected by the mutations. Thus, the alleles that we have isolated of PLE and HYA suggest that the genes may encode organ-specific components needed primarily during root development. Analysis of microtubule arrays during cell cycle in ple and hya roots indicates that the presence of several synchronized nuclei influences the position of preprophase band, mitotic spindles, and phragmoplasts. The enhanced and synergistic phenotype of PLE/ple.hya/hya seedlings and double mutants point to a role of PLE and HYA in the same process. These mutants provide tools to elucidate the regulation of nuclear cytoskeletal interactions during cell division and cytokinesis.

• Weijers D, Franke-van Dijk M, Vencken RJ, Quint A, Hooykaas P, Offringa R
• An Arabidopsis Minute-like phenotype caused by a semi-dominant mutation in a RIBOSOMAL PROTEIN S5 gene.
• Development. 2001; 128: 4289-99
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• Mutations in ribosomal protein (RP) genes in Drosophila lead to strong developmental phenotypes, expressed in the semi-dominant Minute syndrome. In plants, however, mutations in RP genes have so far only been reported to result in recessive developmental phenotypes. We present the analysis of an Arabidopsis promoter-trap line, in which a T-DNA insertion in an RPS5 gene (AtRPS5A) causes semi-dominant developmental phenotypes. Most cell-division processes are delayed or disturbed in the heterozygous mutant, and development is completely arrested at an early embryonic stage in the homozygous mutant. By analogy with Drosophila rp mutants, we have named this mutant Arabidopsis Minute-like 1 (aml1). As with other Arabidopsis RPs, RPS5 is represented by a small gene family, but in contrast to other described plant RPs, this family comprises only two members. The AtRPS5A gene (mutated in aml1) is strongly expressed in dividing cells, whereas expression of the second RPS5 gene, AtRPS5B, is lower than that of AtRPS5A, and is correlated with cell differentiation rather than cell division. From expression analyses we conclude that AtRPS5A is the most abundantly expressed RPS5 gene in Arabidopsis. The Minute-like defects in the aml1 mutant provide the first evidence that ribosome insufficiency leads to similar consequences in both plants and insects, and emphasize the general importance of efficient protein translation for cell proliferation in higher eukaryotes.

• Catterou M et al.
• Brassinosteroids, microtubules and cell elongation in Arabidopsis thaliana. I. Molecular, cellular and physiological characterization of the Arabidopsis bull mutant, defective in the delta 7-sterol-C5-desaturation step leading to brassinosteroid biosynthesis.
• Planta. 2001; 212: 659-72
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• Although cell elongation is a basic function of plant morphogenesis, many of the molecular events involved in this process are still unknown. In this work an extremely dwarf mutant, originally named bul, was used to study one of the main processes of plant development, cell elongation. Genetic analyses revealed that the BUL locus was linked to the nga172 marker on chromosome 3. Recently, after mapping the new dwf7 mutation of Arabidopsis, which is allelic to ste1, it was reported that dwf7 is also linked to the same marker. Sterol analyses of the bull-1 mutant indicated that bul1-1 is defective in the delta 7-sterol-C5-desaturation step leading to brassinosteroid biosynthesis. Considering these findings, we designated our bul mutant as bul1-1/dwf7-3/ste1-4. The bul1-1 mutant was characterized by a very dwarf phenotype, with delayed development and reduced fertility. The mutant leaves had a dark-green colour, which was probably due to continuous stomatal closure. The bul1-1 mutant showed a partially de-etiolated phenotype in the dark. Cellular characterization and rescue experiments with brassinosteroids demonstrated the involvement of the BUL1-1 protein in brassinosteroid-dependent plant growth processes.

• Mimida N et al.
• Functional divergence of the TFL1-like gene family in Arabidopsis revealed by characterization of a novel homologue.
• Genes Cells. 2001; 6: 327-36
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• BACKGROUND: The TERMINAL FLOWER 1 (TFL1) gene of Arabidopsis plays an important role in regulating flowering time and in maintaining the fate of inflorescence meristem (IM). TFL1 is a homologue of CENTRORADIALIS (CEN) from Antirrhinum, which is only involved in IM maintenance. Recent mutational studies and the genome project revealed that TFL1 belongs to a small gene family in Arabidopsis, in which functional divergence may have occurred among the members. RESULTS: We found a new member of the TFL1 gene family, which is mapped on chromosome 2 of Arabidopsis. The predicted protein sequence encoded by this gene is more closely related to that of CEN than other Arabidopsis TFL1 homologues (and therefore named ATC for Arabidopsis thaliana CENTRORADIALIS homologue). Transgenic plants constitutively expressing the ATC gene (35S:ATC), in either wild-type or tfl1 mutant backgrounds, showed a phenotype similar to that observed in transgenic plants constitutively expressing the TFL1 gene. However, in contrast to TFL1, the expression of ATC was only detected in the hypocotyl of young plants, and not in the IM. In addition, an atc loss-of-function mutant, isolated by screening a T-DNA library, showed no phenotypes that were similar to those of tfl1 mutants. CONCLUSION: The phenotypes of transgenic plants over-expressing ATC suggest that the ATC protein can functionally substitute for TFL1. However, the pattern and level of expression and the loss-of-function phenotype indicate that ATC does not participate in the regulation of IM identity, but rather has a role that is different from that of TFL1.

• Han SY, Zhou L, Upadhyaya A, Lee SH, Parker KL, DeJong J
• TFIIAalpha/beta-like factor is encoded by a germ cell-specific gene whose expression is up-regulated with other general transcription factors during spermatogenesis in the mouse.
• Biol Reprod. 2001; 64: 507-17
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• TFIIAalpha/beta-like factor (ALF) is a testis-specific counterpart of the large subunit of human general transcription factor TFIIA. Northern analysis shows that ALF mRNA first appears in mouse testis at Postnatal Day 14. Similarly, expression of the general transcription factors TBP, TRF2, TFIIAalpha/beta, TFIIAgamma, and TFIIIB(90) is also increased beginning at Postnatal Day 14, suggesting that there is a coordinated induction of many general transcription factors during male germ cell differentiation. Analysis of male germ cells separated by Staput sedimentation shows that ALF is present in pachytene spermatocytes and haploid spermatids. In addition, in situ hybridization experiments with adult mouse testis shows that ALF is present in haploid spermatids. Searches of the human genome sequence database using the basic local alignment search tool reveal that the ALF and TFIIAalpha/beta(GTF2A1) genes are both composed of nine exons, whereas the TFIIAgamma (GTF2A2) gene is composed of five exons. Furthermore, nucleotide and amino acid comparisons among human and mouse ALF, TFIIAalpha/beta, and TFIIAgamma cDNA sequences show that ALF has diverged more rapidly than either TFIIAalpha/beta or TFIIAgamma. Finally, the ALF and SBLF (Stoned B-Like Factor) sequences present in the chimeric SALF cDNA are both present on human chromosome 2, and an analysis of the corresponding genes suggests a model for the formation of SALF.

• Song JY, Leung T, Ehler LK, Wang C, Liu Z
• Regulation of meristem organization and cell division by TSO1, an Arabidopsis gene with cysteine-rich repeats.
• Development. 2000; 127: 2207-17
• Display abstract

• In higher plants, meristem organization and cell division regulation are two fundamentally important and intimately related biological processes. Identifying and isolating regulatory genes in these processes is essential for understanding higher plant growth and development. We describe the molecular isolation and analyses of an Arabidopsis gene, TSO1, which regulates both of these processes. We previously showed that tso1 mutants displayed defects in cell division of floral meristem cells including partially formed cell walls, increased DNA content, and multinucleated cells (Liu, Z., Running, M. P. and Meyerowitz, E. M. (1997). Development 124, 665-672). Here, we characterize a second defect of tso1 in influorescence meristem development and show that the enlarged influorescence in tso1 mutants results from repeated division of one inflorescence meristem into two or more influorescence meristems. Using a map-based approach, we isolated the TSO1 gene and found that TSO1 encodes a protein with cysteine-rich repeats bearing similarity to Drosophila Enhancer of zeste and its plant homologs. In situ TSO1 mRNA expression pattern and the nuclear localization of TSO1-GFP are consistent with a regulatory role of TSO1 in floral meristem cell division and in influorescence meristem organization.

• Inoue A et al.
• The transcript for a novel protein with a zinc finger motif is expressed at specific stages of mouse spermatogenesis.
• Biochem Biophys Res Commun. 2000; 273: 398-403
• Display abstract

• The cDNA for an RNA that is expressed predominantly in mouse spermatogenic cells was cloned and characterized. It was found to encode novel zinc finger protein. We first generated a cDNA fragment from mouse osteoblastic cells by the differential display method. To our surprise, Northern blot analysis revealed that the corresponding transcript was expressed at high levels in the testis rather than in osteoblastic cells. Therefore, using this fragment as a probe, we isolated the full-length cDNA (3340 bp) from a mouse testis cDNA library. Analysis of the open reading frame of the cDNA indicated that the encoded protein was a polypeptide of 942 amino acids residues that included three distinct domains, namely, a zinc finger domain of the Cys(2)-His(2) type, four basic amino acid-rich domains, and a myosin II-homology domain. In situ hybridization indicated that the transcript was present in seminiferous tubules of adult mice. Elevated expression of the transcript during testicular development in mice was restricted to spermatocytes at the pachytene stage of meiotic prophase and to round and elongated spermatids, as indicated by Northern blot analysis and RT-PCR. Our results suggest that this novel zinc finger protein might act as a transcriptional regulator during spermatogenesis and, in particular, during meiotic division.

• Deveaux Y, Alonso B, Pierrugues O, Godon C, Kazmaier M
• Molecular cloning and developmental expression of AtGR1, a new growth-related Arabidopsis gene strongly induced by ionizing radiation.
• Radiat Res. 2000; 154: 355-64
• Display abstract

• Screening for mRNAs that accumulate after DNA damage induced by ionizing radiation, we have isolated a 2.0-kb cDNA coding for a new Arabidopsis PEST-box protein named AtGR1 (A. thaliana gamma response 1) with an expression profile similar to that observed for several plant cell cycle-related proteins. Using an anti-AtGR1 antibody, we have shown that the AtGR1 protein is expressed at basal levels in mitotically dividing cells (meristematic tissues and organ primordia) and at a strongly enhanced level in endoreduplicating cells (stipules, trichomes). Using transgenic Arabidopsis plants that express the GUS reporter gene under the control of the AtGR1 promoter, we have demonstrated that the observed AtGR1 protein distribution is due to the promoter activity. Our results suggest that basal AtGR1 levels are associated with progression through mitosis, whereas elevated intracellular levels of AtGR1 seem to induce changes between the S and M phases of the cell cycle that trigger somatic cells to enter the endoreduplication cycle. Ionizing radiation-induced rapid and dose-dependent accumulation of AtGR1 mRNA in cell cultures and plant tissues leads to tissue-specific accumulation of AtGR1 protein, best observed in ovules, which never undergo an endoreduplication cycle. It therefore appears that the radiation-induced transient AtGR1 accumulation reflects DNA damage-dependent transient cell cycle arrest before mitosis, which is necessary to accomplish DNA repair prior to chromosome segregation and cytokinesis.

• Mussig C, Kauschmann A, Clouse SD, Altmann T
• The Arabidopsis PHD-finger protein SHL is required for proper development and fertility.
• Mol Gen Genet. 2000; 264: 363-70
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• The SHL gene from Arabidopsis thaliana encodes a small nuclear protein that contains a BAH domain and a PHD finger. Both domains are found in numerous (putative) transcriptional regulators and chromatin-remodeling factors. Different sets of transgenic lines were established to analyze the physiological relevance of SHL. SHL expression driven by the CaMV 35S promoter results in reduced growth, early flowering, early senescence, and impaired flower and seed formation. Antisense inhibition of SHL expression gives rise to dwarfism and delayed development. In-frame N-terminal fusion of the SHL protein to beta-glucuronidase (GUS) directs GUS to the nucleus of stably transformed Arabidopsis plants. Thus, SHL encodes a novel putative regulator of gene expression, which directly or indirectly influences a broad range of developmental processes.

• Sim DL, Chow VT
• The novel human HUEL (C4orf1) gene maps to chromosome 4p12-p13 and encodes a nuclear protein containing the nuclear receptor interaction motif.
• Genomics. 1999; 59: 224-33
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• A 3250-bp novel human cDNA sequence was isolated from the MRC-5 human embryonic lung cell line by the rapid amplification of cDNA ends technique. This gene was designated HUEL and given the symbol C4orf1 by the HUGO Nomenclature Committee. Within HUEL was identified a continuous ORF of 1704 bp encoding a predicted hydrophilic protein of 568 amino acids with a calculated molecular mass of 63,410 Da. The putative protein contains the LXXLL signature motif considered necessary and sufficient for binding of certain coactivators to liganded nuclear receptors, as well as nuclear localization signals, a nuclear export-like signal, a zinc finger-like motif, an acidic region, and two leucine zipper-like domains. Northern blot analysis of human fetal tissues revealed 3. 4-kb transcripts, while RT-PCR demonstrated HUEL expression in a wide range of human adult tissues and cancer cell lines. In the SiHa, HT-1080, and G-401 cancer lines was detected an alternative transcript in which a 166-bp segment was excluded by exon skipping, which is predicted to culminate in a protein with a modified and truncated C-terminus. HUEL was localized to chromosome region 4p12-p13 by fluorescence in situ hybridization. In Western blots, affinity-purified antibodies raised against a HUEL-specific synthetic peptide could recognize a distinct protein band of approximately 70 kDa. Immunoblotting of subcellular fractions and indirect immunofluorescence of human embryonic lung cells demonstrated the distribution of HUEL predominantly in the cytoplasm, with an apparently cytoskeletal association. However, in smaller or dividing PLC/PRF/5 and TONG liver carcinoma cells, there was a translocation of HUEL from the cytoplasm to the nucleus. Taken together, these data suggest that HUEL plays a role in transcriptional regulation.

• Loong Chan V, Louie H, Joe A
• Expression of the flgFG operon of Campylobacter jejuni in Escherichia coli yields an extra fusion protein.
• Gene. 1998; 225: 131-41
• Display abstract

• Two Campylobacter jejuni genes with homology to the Escherichia coli flgF and flgG genes encoding two of the basal body rod proteins were isolated, and the nucleotide sequence was determined and analyzed. These two C. jejuni genes were shown, by Northern hybridization analysis, to function as a single operon (flgFG). Two transcriptional start sites were detected upstream of flgF, corresponding to the two RNA transcripts detected in the Northern blot. Western blot immunoassays using anti-FlgF and anti-FlgG antibodies demonstrated the synthesis of FlgF and FlgG proteins in C. jejuni and in Escherichia coli containing the C. jejuni flgF and flgG genes. Maxicell analysis and Western immunoblots using anti-FlgF antibodies to probe flgFG-encoded proteins in E. coli revealed the presence of a protein with a molecular mass of approximately the combined mass of the FlgF and FlgG proteins. Anti-FlgF antibodies detected in C. jejuni cell extracts the native FlgF protein and also a higher-molecular-weight protein that is likely encoded by the flgF and part of the flgG sequences.

• Baytel D, Shalom S, Madgar I, Weissenberg R, Don J
• The human Pim-2 proto-oncogene and its testicular expression.
• Biochim Biophys Acta. 1998; 1442: 274-85
• Display abstract

• In this study we describe the cloning of a human gene, encoding a protein that shares 90% identity and 93% similarity at the primary structure level, with the mouse Pim-2 gene. The gene was designated hPim-2. Structural features suggest that like the mouse Pim-2, hPim-2 is also a serine threonine kinase. At the RNA level, two hPim-2 transcripts were identified. The first, 2.2 kb, is highly expressed in hematopoietic tissues and in leukemic and lymphoma cell lines (K-562, HL-60 and RAJI). It also shows considerable high levels in testis, small intestine, colon and human colorectal adenocarcinoma cells (SW480). A second transcript, 5.0 kb in size, could be detected only in spleen, thymus, small intestine and colon and in the K-562 and RAJI cell lines. In situ hybridization analysis of biopsies taken from testes of men with complete or partial spermatogenesis revealed that the gene is expressed in primary spermatocytes. In the absence of germ cells, signal could be detected over specific cells in the well developed interstitial region. These results suggest a role for hPim-2 in proliferating cells as well as during meiosis. A possible connection between hPim-2 and apoptosis is discussed.

• Takahashi H, Koshimizu U, Nakamura T
• A novel transcript encoding truncated LIM kinase 2 is specifically expressed in male germ cells undergoing meiosis.
• Biochem Biophys Res Commun. 1998; 249: 138-45
• Display abstract

• LIM kinases, composed of LIMK1 and LIMK2, have unique structural features that contain two LIM motifs at the N-terminus and a catalytic domain at the C-terminus. We report evidence of a novel type of mouse LIMK2 (Limk2) transcript specifically expressed in testis. cDNA cloning showed this Limk2 variant, designated tLimk2, lacked LIM domains at the N-terminus, due to usage of a testis-specific, alternative initiation exon. In Northern blot analysis, tLimk2 was detected in intact adult testis, but not in germ-cell-deficient or immature testis, indicating the stage-specific expression of tLimk2 in spermatogenic cells. In situ hybridization clearly demonstrated that tLimk2 was restrictedly expressed in differentiated germ cells (pachytene spermatocytes to round spermatids) and not expressed in early stages of spermatogenic cells and somatic cells in testis. These results suggested the possibility that the tLimk2 product is involved in spermatogenesis, especially in meiotic and/or postmeiotic processes.

• Pao SS, Paulsen IT, Saier MH Jr
• Major facilitator superfamily.
• Microbiol Mol Biol Rev. 1998; 62: 1-34
• Display abstract

• The major facilitator superfamily (MFS) is one of the two largest families of membrane transporters found on Earth. It is present ubiquitously in bacteria, archaea, and eukarya and includes members that can function by solute uniport, solute/cation symport, solute/cation antiport and/or solute/solute antiport with inwardly and/or outwardly directed polarity. All homologous MFS protein sequences in the public databases as of January 1997 were identified on the basis of sequence similarity and shown to be homologous. Phylogenetic analyses revealed the occurrence of 17 distinct families within the MFS, each of which generally transports a single class of compounds. Compounds transported by MFS permeases include simple sugars, oligosaccharides, inositols, drugs, amino acids, nucleosides, organophosphate esters, Krebs cycle metabolites, and a large variety of organic and inorganic anions and cations. Protein members of some MFS families are found exclusively in bacteria or in eukaryotes, but others are found in bacteria, archaea, and eukaryotes. All permeases of the MFS possess either 12 or 14 putative or established transmembrane alpha-helical spanners, and evidence is presented substantiating the proposal that an internal tandem gene duplication event gave rise to a primordial MFS protein prior to divergence of the family members. All 17 families are shown to exhibit the common feature of a well-conserved motif present between transmembrane spanners 2 and 3. The analyses reported serve to characterize one of the largest and most diverse families of transport proteins found in living organisms.

• Chen L, Sato M, Inoko H, Kimura M
• Molecular cloning and analysis of novel cDNAs specifically expressed in adult mouse testes.
• Biochem Biophys Res Commun. 1997; 240: 261-8
• Display abstract

• In an effort to examine the molecular basis of spermatogenesis, we isolated two types of novel cDNA clones specifically expressed in mouse testes. Type A cDNA (2,071 nucleotides) is predicted to encode 347 amino acid residues, whereas type B cDNA (1,536 nucleotides) has a deletion of 535 bp from nucleotides 1,206 to 1,740 of type A cDNA, probably due to alternative splicing. This deletion causes a frame shift of the putative open reading frame at the C-terminal portion of type A cDNA to encode 366 amino acid residues. Northern blot analysis using adult ICR organs demonstrated that both types of mRNAs were specifically expressed in testis, although type B mRNA was more abundant than type A mRNA. RT-PCR analysis revealed that these two mRNAs were also expressed in immature testes at 1, 5, 11, 16 and 24 days after birth. In situ hybridization analysis of adult ICR testes demonstrated that these two mRNAs were expressed in spermatogonia, Sertoli cells and Leydig cells. In the W/ WV mouse testis which lacks c-kit activity and spermatogonia, but contains Sertoli and Leydig cells, both mRNAs were found to be expressed in the latter two types of cells. We therefore termed these novel clones tsec-1, testis-specifically expressed cDNAs-1. The protein products of tsec-1 may play an important role in mammalian spermatogenesis.

• Jones MH et al.
• Chromosomal assignment of 311 sequences transcribed in human adult testis.
• Genomics. 1997; 40: 155-67
• Display abstract

• A total of 311 expressed sequence tags (ESTs) derived from human adult testis have been assigned to human chromosomes by Southern analysis of a monochromosome somatic cell hybrid panel. Over 70% of the ESTs show conservation to hamster and mouse DNA, and the overall distribution of transcripts correlates well with physical chromosome size and to a greater extent with male meiotic chromosome length. The notable exception is the X chromosome, for which the number of testis-derived ESTs is greatly underrepresented. This finding may reflect inactivation of the X chromosome during the meiotic phase of spermatogenesis and a consequent selection against large numbers of X-linked germ cell transcripts. Further analysis of the distribution of testis ESTs showed that the EST density remains significantly correlated with the recombination density of each autosome. Analysis of a comparable number (320) of brain EST autosome assignments showed no similar correlation. These data suggest a specific association between transcription in testis tissue and male meiotic recombination.

• Voss GC, Jockusch H
• New testis-specific expressed genes on mouse Chromosome 11.
• Mamm Genome. 1996; 7: 163-163

• Jin YH et al.
• Characterization of SFP2, a putative sulfate permease gene of Saccharomyces cerevisiae.
• Biochem Biophys Res Commun. 1995; 214: 709-15
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• The SFP2 gene of Saccharomyces cerevisiae has been characterized. The deduced amino acid sequence contained twelve highly hydrophobic domains and showed 50, 47, 44 and 48% homologies to Neurospora crassa sulfate permease II (CYS14), soybean GMAK170 nodulin, human colon mucosa protein (DRA) and a putative open reading frame (ORF) downstream of Escherichia coli prs (phosphoribosyl pyrophosphatate synthetase) gene, respectively, in the aligned regions. Cells lacking SFP2 were viable and displayed no obvious decrease in their growth rate. Southern blot analysis revealed that SFP2 exists as a single copy in haploid genome. Northern blot analysis showed that SFP2 produced a 2.8-kb transcript which was highly expressed under sulfur derepressing condition. SFP2 mRNA was found to turn over with a half-life of approximately 15 min, which may contribute to the regulation of sulfate permease function, and reached its maximal level in about 22 h after depression.

• Koh YS, Roe JH
• Isolation of a novel paraquat-inducible (pqi) gene regulated by the soxRS locus in Escherichia coli.
• J Bacteriol. 1995; 177: 2673-8
• Display abstract

• We have isolated promoters inducible by paraquat, a superoxide radical-generating agent, from Escherichia coli, using promoter-probing plasmid pJAC4 (Y.S. Koh and J.H. Roe, Korean J. Microbiol. 31:267-273, 1993). One promoter clone pqi-5 (pqi denotes paraquat-inducible gene) was mapped at 21.8 min on the E. coli chromosome by using the Kohara phage library. We constructed an operon fusion of the lacZ gene with the pqi-5 promoter to monitor the expression of the gene in the single-copy state. LacZ expression was induced about 7- to 13-fold by 77 to 780 microM paraquat. Other known superoxide generators such as menadione, phenazine methosulfate, and plumbagin also induced the expression of beta-galactosidase in this fusion strain. On the other hand, no significant induction was observed with treatment with hydrogen peroxide, ethanol, and heat shock. Induction of beta-galactosidase was significantly reduced by introducing a delta sox-8::cat or soxS3::Tn10 mutation into the fusion strain, indicating that pqi-5 is a member of the soxRS regulon. A DNA fragment containing the pqi-5 promoter was cloned and sequenced from the Kohara phage E2E5. We identified two pqi-5 open reading frames (ORFs); ORF-A encodes a predicted protein of 342 amino acids, and ORF-B is truncated at the cloning site. The transcription start site from the pqi-5 promoter was determined by primer extension and S1 nuclease protection analyses. Northern (RNA) and S1 analyses indicated that there are two kinds of pqi-5 transcript; one covers ORF-A only and the other covers ORF-A and possibly also ORF-B.

• Lobocka M, Hennig J, Wild J, Klopotowski T
• Organization and expression of the Escherichia coli K-12 dad operon encoding the smaller subunit of D-amino acid dehydrogenase and the catabolic alanine racemase.
• J Bacteriol. 1994; 176: 1500-10
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• A fragment of the Escherichia coli K-12 chromosome complementing the D-amino acid dehydrogenase and catabolic alanine racemase deficiency of a dad operon deletion mutant was cloned in a mini-Mu plasmid. The dadA and dadX genes were localized to a 3.5-kb part of the plasmid insert. The nucleotide sequence of this fragment revealed two open reading frames encoding 432- and 356-amino-acid-long proteins. We show here that they correspond to the dadA and dadX genes. The dadA gene can encode only the smaller of the two subunits of D-amino acid dehydrogenase. A computer search revealed the presence of a flavin adenine dinucleotide-binding motif in the N-terminal domain of the deduced DadA protein sequence. This is in agreement with biochemical data showing that the D-amino acid dehydrogenase contains flavin adenine dinucleotide in its active center. The predicted dadX gene product appeared to be 85% identical to a dadB-encoded catabolic alanine racemase of Salmonella typhimurium. The organization of the dadA and dadX genes confirmed our previous conclusion based on the genetic data (J. Wild, J. Hennig, M. Lobocka, W. Walczak, and T. Klopotowski, Mol. Gen. Genet. 198:315-322, 1985) that these genes form an operon. The main transcription start points of the dad operon were determined by primer extension. They are preceded by a putative sigma 70 promoter sequence and two cyclic AMP-cyclic AMP receptor protein (cAMP-CRP) binding sites, one of higher and one of lower affinity to CRP. We propose that the high-affinity site, centered 59.5 bp upstream of the main transcription start point, plays a role in cAMP-CRP-mediated activation of dad operon expression in the absence of glucose.

• Hussain H, Grove J, Griffiths L, Busby S, Cole J
• A seven-gene operon essential for formate-dependent nitrite reduction to ammonia by enteric bacteria.
• Mol Microbiol. 1994; 12: 153-63
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• The DNA sequence of the regulatory region and the structural gene, nrfA, for cytochrome c552 of Escherichia coli K-12 have been reported. We have now established that nrfA is the first gene in a seven-gene operon, designated the nrf operon, at least five of which are essential for formate-dependent nitrite reduction to ammonia. This operon terminates just upstream of the previously sequenced gltP gene encoding a sodium-independent, glutamate and aspartate transporter. Expression of lac fused to nrfA, nrfE or nrfG is regulated by oxygen repression, FNR-dependent anaerobic induction, nitrite induction and nitrate repression during anaerobic growth, exactly as previously reported for the nrfA promoter. In contrast, expression of the gltP-lac fusion was FNR-independent. The open reading frame immediately downstream of nrfA encodes NrfB, a hydrophilic, penta-haem cytochrome c with an M(r) of 20,714. The structure of the N-terminal region is typical of a signal peptide for a periplasmic protein: cleavage at the putative signal peptide cleavage site, Ala-26, would result in a periplasmic cytochrome with a molecular mass of 18 kDa. The NrfC polypeptide, M(r) 24,567, contains 16 cysteine residues arranged in four clusters typical of the CooF super-family of non-haem iron-sulphur proteins. The NrfD sequence predicts a 318-residue hydrophobic protein with a distribution of acidic and basic amino acids which suggests that NrfD is an integral transmembrane protein with loops in both the periplasm and the cytoplasm. Proteins most similar to NrfD include the PsrC subunit of polysulphide reductase from Wolinella, but, as seven of the 10 most similar proteins are NADH-ubiquinone oxidoreductases, we propose that NrfD participates in the transfer of electrons from the quinone pool into the terminal components of the Nrf pathway. NrfE, M(r) 60,851, is predicted to be another hydrophobic, integral membrane protein homologous to the CdI1 protein of Rhodobacter capsulatus, which has been implicated in the assembly of periplasmic c-type cytochromes. The sequence of the 127 residue NrfF polypeptide, M(r) 14,522, is strikingly similar to the CcI2 protein of R. capsulatus, especially in the putative haem-binding motif, RCPQCQNQN.(ABSTRACT TRUNCATED AT 400 WORDS)

• Chen NY, Jiang SQ, Klein DA, Paulus H
• Organization and nucleotide sequence of the Bacillus subtilis diaminopimelate operon, a cluster of genes encoding the first three enzymes of diaminopimelate synthesis and dipicolinate synthase.
• J Biol Chem. 1993; 268: 9448-65
• Display abstract

• The nucleotide sequence of a 7-kilobase segment of the Bacillus subtilis chromosome containing the entire coding regions for the enzymes catalyzing the first three steps of diaminopimelate synthesis as well as dipicolinate synthase has been determined. This group of functionally related genes, termed the dap operon, were arranged in the order orfY, orfX, asd, dapG, and dapA and were bracketed by potential rho-independent transcription terminators. The asd locus could complement the growth defect of Escherichia coli strains with an asd deletion. Disruption of the dapG locus led to the loss of aspartokinase I, with a phenotype similar to that of the temperature-sensitive dapG mutants described earlier (Roten, C. A. H., Brandt, C., and Karamata, D. (1991) J. Gen. Microbiol. 137, 951-962). The amino acid sequences of the deduced products of the asd, dapG, and dapA loci had high degrees of similarity with those of other aspartate semialdehyde dehydrogenases, aspartokinases, and dihydrodipicolinate synthases, respectively. Disruption of orfX had no effect on growth but caused a sporulation defect, characterized by low sporulation frequencies and heat-sensitive spores, which could be cured by supplementation with dipicolinate, similar to the phenotype of mutants defective in spoVF, the putative structural gene for dipicolinate synthase. Two other open reading frames, upstream of spoVF, encoded the 380 COOH-terminal residues of a protein homologous to mitochondrial processing proteases and an 85-residue polypeptide of unknown function. Transcription initiation sites associated with the orfY-orfX-asd-dapG-dapA gene cluster were mapped by primer extension. The results indicate that during vegetative growth, the three distal genes of the dap operon, asd, dapG, and dapA, are transcribed as a unit and orfY and orfX are not expressed, whereas at stage 5 of sporulation two separate transcripts are produced, one comprising all five genes, the other just the three distal genes of the operon.

• Cohen SP, Hachler H, Levy SB
• Genetic and functional analysis of the multiple antibiotic resistance (mar) locus in Escherichia coli.
• J Bacteriol. 1993; 175: 1484-92
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• A 7.8-kbp fragment of chromosomal DNA from a region controlling multiple antibiotic resistance (Mar) in Escherichia coli has been sequenced. Within the fragment is a potential divergent promoter region including marO, which contains two pairs of direct repeats, suggesting possible operator-regulatory sites. To the left of marO (region I) are one or two transcriptional units with three putative open reading frames (ORFs) encoding 64, 157, and 70 amino acids. To the right (region II) is a transcriptional unit containing three putative ORFs (ORF125/144, ORF129, and ORF72). Of six independent Mar mutants, four had mutations within the ORF encoding the first putative protein (ORF125/144) downstream of marO, including three different single-point mutations and an IS2 insertion. One of the other mutations occurred in marO (20-bp duplication), and the other occurred in a site in marO or ORF144 (a 1-bp change). All six mutations led to increased transcription of the region II transcript. High-copy-number plasmids containing marO and the adjacent ORF125/144 region from a wild-type source but not from a Mar mutant reduced the antibiotic resistance of a Mar mutant to levels comparable to those of wild-type cells. High-copy-number plasmids containing wild-type marO alone caused an increase in resistance to tetracycline, chloramphenicol, and norfloxacin in a wild-type strain. The nature of the Mar mutations and the results of the complementation studies suggest that ORF125/144 encodes a repressor (designated MarR) which acts at marO. The second ORF (ORF129), designated marA, would encode a protein, MarA, whose sequence shows strong similarity to those of a family of positive transcriptional regulators. A Tn5 insertion in marA inactivated the multiresistance phenotype of Mar mutants. The function of ORF72, designated marB, encoding the third putative protein in the operon, and that of other ORFs detected within the 7.8-kb fragment have not yet been determined.

• Thomson VJ, Jovanovic OS, Pohlman RF, Chang CH, Figurski DH
• Structure, function, and regulation of the kilB locus of promiscuous plasmid RK2.
• J Bacteriol. 1993; 175: 2423-35
• Display abstract

• The kil-kor regulon of the self-transmissible, broad-host-range plasmid RK2 is a unique network with eight coregulated operons. Among the genes encoded by the kil-kor regulon are trfA, which encodes the replication initiator, and several kil loci (kilA, kilB, kilC, and kilE), each of which is lethal to the host cell in the absence of appropriate negative regulatory elements encoded by the korA, korB, korC, and korE determinants. We have proposed that the functions of the kil loci are related to RK2 maintenance or host range. Here, we report the nucleotide sequence of a 2.44-kb region that includes the lethal kilB determinant. We identified the first three genes of the kilB operon (designated klbA, klbB, and klbC), and we determined by deletion analysis that the host-lethal phenotype requires klbB. The predicted amino acid sequence of the 34,995-Da klbA product reveals a potential ATP-binding fold. The klbB product is predicted to be a membrane protein with a molecular mass of 15,012 Da with homology to the RK2 KlaC membrane protein encoded by the kilA operon. The amino acid sequence of the 12,085-Da klbC product contains a perfect match to the leucine zipper motif common to eukaryotic regulatory proteins. Primer extension analysis revealed unambiguously that transcription of the kilB operon begins 46 nucleotides upstream of klbA. No transcription was initiated from the sequence previously presumed by other investigators to be the kilB promoter. The abundance of kilB transcripts is reduced in the presence of KorB, consistent with the prediction that KorB acts at the level of transcription. A degenerate KorB-binding site that contains a perfect half-palindrome overlaps the kilB promoter, but this site is insufficient for regulation by KorB. The region containing a KorB-binding site located 183 bp upstream of the transcriptional start is required for regulation by KorB, indicating that KorB acts at a distance to regulate transcription of kilB. Our studies with the mutant plasmid pRP101, a transfer-defective derivative of the RK2-like plasmid RP4, demonstrated that the kilB operon includes the conjugal transfer and surface exclusion genes of the Tra2 region. Nucleotide sequence analysis revealed that the transposon Tn7 insertion in pRP101 is located in the klbC gene, and complementation analysis showed that this mutation has a strong polar effect on the expression of genes for conjugal transfer and surface exclusion located several kilobases downstream. A klbA mutant was constructed and found to be both transfer defective and complementable, thus, demonstrating a requirement was constructed and found to be both transfer defective and complementable, thus demonstrating a requirement for klbA product in plasmid transmissibility. These results have demonstrated a role for the kilB operon in conjugal transfer. The kil-kor regulon of RK2 is the only known example of plasmid-mediated coregulation of replication and transfer.

• Kuipers OP, Beerthuyzen MM, Siezen RJ, De Vos WM
• Characterization of the nisin gene cluster nisABTCIPR of Lactococcus lactis. Requirement of expression of the nisA and nisI genes for development of immunity.
• Eur J Biochem. 1993; 216: 281-91
• Display abstract

• The nisin gene cluster nisABTCIPR of Lactococcus lactis, located on a 10-kbp DNA fragment of the nisin-sucrose transposon Tn5276, was characterized. This fragment was previously shown to direct nisin-A biosynthesis and to contain the nisP and nisR genes, encoding a nisin leader peptidase and a positive regulator, respectively [van der Meer, J. R., Polman, J., Beerthuyzen, M. M., Siezen, R. J., Kuipers, O. P. & de Vos, W. M. (1993) J. Bacteriol. 175, 2578-2588]. Further sequence analysis revealed the presence of four open-reading frames, nisB, nisT, nisC and nisI, downstream of the structural gene nisA. The nisT, nisC and nisI genes were subcloned and expressed individually in Escherichia coli, using the T7-RNA-polymerase system. This resulted in the production of radiolabelled proteins with sizes of 45 kDa (NisC) and 32 kDa (NisI). The nisT gene product was not detected, possibly because of protein instability. The deduced amino acid sequence of NisI contained a consensus lipoprotein signal sequence, suggesting that this protein is a lipid-modified extracellular membrane-anchored protein. Expression of nisI in L. lactis provided the cells with a significant level of protection against exogenously added nisin, indicating that NisI plays a role in the immunity mechanism. In EDTA-treated E. coli cells, expression of nisI conferred up to a 170-fold increase in immunity against nisin A compared to controls. Moreover, a lactococcal strain deficient in nisin-A production, designated NZ9800, was created by gene replacement of nisA by a truncated nisA gene and was 10-fold less resistant to nisin A than the wild-type strain. A wild-type immunity level to nisin and production of nisin was obtained in strain NZ9800 harboring complementing nisA and nisZ plasmids. Transcription analyses of several L. lactis strains indicated that an expression product of the nisA gene, together with NisR, is required for the activation of nisA transcription.

• de Boer PA
• Chromosome segregation and cytokinesis in bacteria.
• Curr Opin Cell Biol. 1993; 5: 232-7
• Display abstract

• Substantial progress has recently been made in the understanding of chromosome partitioning and cytokinesis in bacteria. The biochemical properties of some key protein components involved in these processes are beginning to emerge. New evidence supports the recently developed notion that, in prokaryotic cells, basic cell biological processes rely on the activity of previously unidentified cytoskeletal-like elements.

• Yoshida T, Ueguchi C, Yamada H, Mizuno T
• Function of the Escherichia coli nucleoid protein, H-NS: molecular analysis of a subset of proteins whose expression is enhanced in a hns deletion mutant.
• Mol Gen Genet. 1993; 237: 113-22
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• The expression of numerous Escherichia coli cellular proteins was previously demonstrated to be greatly enhanced in a hns deletion background, relative to the levels in wild-type cells. In this study, a subset of such proteins, expression of which is affected by H-NS, was partially purified, and the genes coding for some of the proteins were identified and characterized. Two of the proteins thus characterized, 19K and 17K, were found to be encoded by previously predicted genes that are located adjacent to, and downstream of, the trpABCDE operon (27.6 min on the E. coli genetic map). The genes coding for the other two proteins, 10K-L and 10K-S, are located at 77.5 min on the genetic map. Their nucleotide sequences were determined and revealed that they may constitute an operon. To characterize the putative promoters for these genes, a set of promoter-lacZ transcriptional fusion genes was constructed on the E. coli chromosome. The results of such promoter-probe analyses indicated that H-NS represses the expression of these genes at the transcriptional level. Furthermore, H-NS appeared to exhibit relatively strong affinity for the putative promoter sequences in vitro. These results are compatible with the hypothesis that H-NS functions as a transcriptional repressor.

• Danielsen S, Kilstrup M, Barilla K, Jochimsen B, Neuhard J
• Characterization of the Escherichia coli codBA operon encoding cytosine permease and cytosine deaminase.
• Mol Microbiol. 1992; 6: 1335-44
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• The nucleotide sequence of a 3.1 kb segment carrying the cytosine deaminase gene (codA) from Escherichia coli was determined. The sequence revealed the presence of two open reading frames, the first (codB) specifying a highly hydrophobic polypeptide and the second specifying cytosine deaminase. A two-codon overlap between the two reading frames indicates that they constitute an operon. Transcription of the operon was found to be regulated by exogenous purines. Polypeptides specified by each of the two reading frames were expressed in minicells, and the codB gene product was found to be highly enriched in the membrane fraction. Uptake experiments showed that the CodB protein is required for cytosine transport into the cell and that the intracellular accumulation of cytosine correlated with the codB gene dose. A topological model for the cytosine permease in the cytoplasmic membrane is proposed.

• Sung YC, Fuchs JA
• The Escherichia coli K-12 cyn operon is positively regulated by a member of the lysR family.
• J Bacteriol. 1992; 174: 3645-50
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• A regulatory gene, cynR, was found to be located next to the cyn operon but transcribed in the opposite direction. cynR encodes a positive regulatory protein that controls the cyn operon as well as its own synthesis. Positive regulation of the cyn operon requires cyanate and the cynR protein, but the negative autoregulation of the cynR gene appears to be independent of cyanate. The predicted amino acid sequence of the cynR protein derived from the DNA sequence was found to have significant homology to the predicted amino acid sequence of the lysR family of regulatory proteins.

• Murakawa GJ, Kwan C, Yamashita J, Nierlich DP
• Transcription and decay of the lac messenger: role of an intergenic terminator.
• J Bacteriol. 1991; 173: 28-36
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• Prior work has indicated that the polycistronic lacZYA mRNA of Escherichia coli is cleaved during decay at approximately intergenic sites (L. W. Lim and D. Kennell, J. Mol. Biol. 135: 369-390, 1979). In this work, we characterized the products by using probes specific for the different cistrons. This analysis indicated that six lac mRNA species are present in the following order of decreasing abundance: lacZ, -A, -ZYA, -ZY, -YA, and -Y. Very little lacYA and lacY mRNAs were present, whereas in cells induced to steady state, there was 10 times more lacZ than lacZYA mRNA. The lacZ mRNA appeared as a discrete species extending to a site in the lacZ-Y intergenic space (ca. residue 3150). This site is just distal to a potential rho-independent termination sequence. We examined the function of this sequence to determine whether it contributes to the distribution of the mRNAs. Although the termination sequence was shown to function in vitro, when it was recloned into an expression vector, no termination was seen in vivo. Moreover, direct examination of the kinetics of lac messenger synthesis revealed that after initiation, most transcription continued to the end of the operon. We conclude that during normal growth, the operon is transcribed in its entirety and that the individual lac mRNAs are formed by cleavage. These results confirm earlier work implying that the lac operon is transcribed in its entirety but are in conflict with several recent reports suggesting that internal termination occurs. Our findings indicate that the natural polarity of the operon (lacZ is expressed sixfold more strongly than lacA) is based on posttranslational effects and not on polarity of transcription.

• Nakahigashi K, Inokuchi H
• Nucleotide sequence between the fadB gene and the rrnA operon from Escherichia coli.
• Nucleic Acids Res. 1990; 18: 6439-6439

• Schweizer HP, Datta P
• The complete nucleotide sequence of the tdc region of Escherichia coli.
• Nucleic Acids Res. 1989; 17: 3994-3994

• Carlomagno MS, Chiariotti L, Alifano P, Nappo AG, Bruni CB
• Structure and function of the Salmonella typhimurium and Escherichia coli K-12 histidine operons.
• J Mol Biol. 1988; 203: 585-606
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• We have determined the complete nucleotide sequence of the histidine operons of Escherichia coli and of Salmonella typhimurium. This structural information enabled us to investigate the expression and organization of the histidine operon. The proteins coded by each of the putative histidine cistrons were identified by subcloning appropriate DNA fragments and by analyzing the polypeptides synthesized in minicells. A structural comparison of the gene products was performed. The histidine messenger RNA molecules produced in vivo and the internal transcription initiation sites were identified by Northern blot analysis and S1 nuclease mapping. A comparative analysis of the different transcriptional and translational control elements within the two operons reveals a remarkable preservation for most of them except for the intercistronic region between the first (hisG) and second (hisD) structural genes and for the rho-independent terminator of transcription at the end of the operon. Overall, the operon structure is very compact and its expression appears to be regulated at several levels.

• Barcellini-Couget S, Galucci P, Bouche JP
• Escherichia coli dicB operon includes a truncated IS2 element.
• Nucleic Acids Res. 1988; 16: 10388-10388

• Overduin P, Boos W, Tommassen J
• Nucleotide sequence of the ugp genes of Escherichia coli K-12: homology to the maltose system.
• Mol Microbiol. 1988; 2: 767-75
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• The nucleotide sequence of the ugp genes of Escherichia coli K-12, which encode a phosphate-limitation inducible uptake system for sn-glycerol-3-phosphate and glycerophosphoryl diesters, was determined. The genetic organization of the operon differed from previously published results. A single promoter, containing a putative pho box, was detected by S1-nuclease mapping. The promoter is followed by four open reading frames, designated ugpB, A, E and C, which encode a periplasmic binding protein, two hydrophobic membrane proteins and a protein that is likely to couple energy to the transport system, respectively. The sequences of the proteins contain the characteristics of several other binding protein-dependent transport systems, but they seem to be particularly closely related to the maltose system.

• Cam K, Bejar S, Gil D, Bouche JP
• Identification and sequence of gene dicB: translation of the division inhibitor from an in-phase internal start.
• Nucleic Acids Res. 1988; 16: 6327-38
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• The dicA1 mutation, located in the replication termination region of Escherichia coli at 34.9 min, confers a temperature-sensitive, division defective phenotype to its hosts. Previous analysis had suggested that dicA codes for a repressor of a nearby division inhibition gene dicB. We show now that gene dicB is part of a complex operon. Five open reading frames (ORFs 1 to 5) preceeded by a promoter sensitive to dicA repression are found within a 1500 bp segment, and are organized into two clusters separated by a long untranslated region. Evidence for expression of these ORFs was obtained from in vitro or in vivo translation of plasmid-coded genes. IPTG-dependent cell filamentation was obtained when either the entire or the C-terminal part of the fourth ORF was placed under control of the lac promoter. In both cases, a 7 KD protein corresponding to translation from an in-frame ATG of ORF4 (dicB) was made. We propose that this C-terminal protein is the division inhibitor synthesized in dicA1 mutants.

• Sung YC, Anderson PM, Fuchs JA
• Characterization of high-level expression and sequencing of the Escherichia coli K-12 cynS gene encoding cyanase.
• J Bacteriol. 1987; 169: 5224-30
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• Restriction fragments containing the gene encoding cyanase, cynS, without its transcriptional regulatory sequences were placed downstream of lac and tac promoters in various pUC derivatives to maximize production of cyanase. Plasmid pSJ105, which contains the cynS gene and an upstream open reading frame, gave the highest expression of cyanase. Approximately 50% of the total soluble protein in stationary-phase cultures of a lac-deleted strain containing plasmid pSJ105 was cyanase. The inserted DNA fragment of pSJ105 was transferred into pUC18 derivatives that contain a hybrid tac promoter, instead of the lac promoter, and a strong terminator to generate pSJ124. Stationary-phase cultures of JM101 containing plasmid pSJ124 overexpressed a similar level of cyanase. In JM101(pSJ124), maximum production of cyanase could be obtained either by induction with isopropyl-beta-D-thiogalactopyranoside (IPTG) for 3 h or by growth without IPTG into late stationary phase. The latter conditions resulted in a 10- to 20-fold increase in plasmid content and presumably titration of the lac repressor. The nucleotide sequence of the cloned cynS gene from Escherichia coli K-12 was determined. The predicted amino acid sequence differed from the known amino acid sequence of cyanase isolated from a B strain by four residues. However, overexpressed cyanase was purified to homogeneity, and a comparison of the enzymes from the two sources indicated that they did not differ with respect to physical and kinetic properties. The cynS gene was located next to the lac operon, and the direction of cynS transcription was opposite that of lac.

• Nielsen J, Jorgensen BB, van Meyenburg KV, Hansen FG
• The promoters of the atp operon of Escherichia coli K12.
• Mol Gen Genet. 1984; 193: 64-71
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• The nucleotide sequence has been determined of a 900 bp segment of chromosomal DNA located between 2.6 and 3.5 kb left of the origin of replication, oriC. This segment, which overlaps with the known sequence of the atp operon coding for the eight subunits of the Escherichia coli K12 ATP synthase, contains two coding sequences with the same polarity (counterclockwise) as the atp genes: One of these, designated atpI, which codes for the N-terminal part of a 14 kD polypeptide, is located in front (upstream) of the atpB gene (the first structural gene in the atp operon), the other one codes for the C-terminal part of the gidB gene. The 606 bp segment located between the gidB and the atpI genes contains no coding sequences. By employing the nuclease S1 mapping technique, we have determined a promoter, designated atpIp, for the atp operon located in front of the atpI gene; two additional, weak transcription starts were located within the atpI gene. No transcription start sites were detected up to 1,000 bp upstream of the atpIp promoter, neither were any transcription start sites detected within the cluster of the eight structural atp genes. The atp operon transcription terminates at a site approximately 50 bp downstream from the atpC gene.

• van Sluis CA, Moolenaar GF, Backendorf C
• Regulation of the uvrC gene of Escherichia coli K12: localization and characterization of a damage-inducible promoter.
• EMBO J. 1983; 2: 2313-8
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• Operon fusion and S1 nuclease mapping have been employed to locate a putative uvrC promoter, which is situated approximately 200 bp ahead of the uvrC structural gene. The promoter sustains transcription towards the uvrC coding sequence and is inducible by DNA damaging agents. The inducibility is dependent on the Escherichia coli LexA and RecA functions. Examination of the DNA sequence in the promoter region reveals the presence of a sequence similar to the consensus of a SOS box. In contrast to the related uvrA and uvrB genes, uvrC gene expression is characterized by a delayed onset of induction after DNA damaging treatment. Furthermore, no induction is observed with nalidixic acid.

• Buchel DE, Gronenborn B, Muller-Hill B
• Sequence of the lactose permease gene.
• Nature. 1980; 283: 541-5
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• The nucleotide sequence of the lacY gene coding for lactose permease (M protein) in Escherichia coli has been determined. The sequence includes the intergenic regions between the lacZ (beta-galactosidase) and lacY genes as well as the region between the lacY and lacA (transacetylase) genes. Lactose permease is predicted to consist of 417 residues (71% nonpolar), resulting in a protein with a molecular weight of 46,504. The reading frame was confirmed by the sequence of a nonsense mutation changing codon 33 from UGG to UAG.

• Adams CW, Lawther RP, Hatfield GW
• The ilvEDA operon of Escherichia coli K12 encodes only one valine-alpha-ketoglutarate transaminase activity.
• Biochem Biophys Res Commun. 1979; 89: 650-8

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