NORMAL GENOMIC

• Vaccaro MC et al.
• Primary structure and developmental expression of Dp ZP2, a vitellineenvelope glycoprotein homolog of mouse ZP2, in Discoglossus pictus, one ofthe oldest living Anuran species.
• Mol Reprod Dev. 2001; 59: 133-43
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• A glycoprotein of the Xenopus vitelline envelope, gp 69/64, which mediatessperm binding, is closely related to the components of ZPA family, such asthe mouse zona pellucida ZP2. To test the generality of these findings, westudied Discoglossus pictus, a species evolutionary distant from Xenopusand identified as a protein of 63 kDa in the vitelline envelope.Preliminary studies suggest that this protein may bind sperm atfertilization. We found that the 63-kDa protein is glycosylated andcontains both N- and O-linked chains. We have cloned the cDNA encoding theDiscoglossus protein of 63 kDa (Dp ZP2) by screening a Discoglossus cDNAlibrary using Xenopus gp 69/64 cDNA as a probe. Analysis of the deducedsequence of Discoglossus protein revealed 48% identity with Xenopus gp69/64 and 37-40% identity with mouse ZP2. The sequence conservationincluded a ZP domain, a potential furin cleavage site and a putativetransmembrane domain. The N-terminus region of Dp ZP2 was 40% identical tothe corresponding region of Xenopus gp 69/64 which has been shown to beessential for sperm binding to the VE. Although, as of yet, there is noevidence for sperm binding at the Dp ZP2 N-terminus, it is interestingthat in this region three potential O-glycosylation sites are conserved inboth species, in contrast to N-glycosylation sites. It was found that theDp ZP2 mRNA is expressed in stage 1 oocytes and in the follicle cellssurrounding the oocyte. Similarly, in Xenopus oocytes, the gp 69/64m RNA,was found in the oocytes, as well as in the somatic cells. Mol. Reprod.Dev. 59:133-143, 2001.

• Becher P, Orlich M, Thiel HJ
• Ribosomal S27a coding sequences upstream of ubiquitin coding sequences inthe genome of a pestivirus.
• J Virol. 1998; 72: 8697-704
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• Molecular characterization of cytopathogenic (cp) bovine viral diarrheavirus (BVDV) strain CP Rit, a temperature-sensitive strain widely used forvaccination, revealed that the viral genomic RNA is about 15.2 kb long,which is about 2.9 kb longer than the one of noncytopathogenic (noncp)BVDV strains. Molecular cloning and nucleotide sequencing of parts of thegenome resulted in the identification of a duplication of the genomicregion encoding nonstructural proteins NS3, NS4A, and part of NS4B. Inaddition, a nonviral sequence was found directly upstream of the secondcopy of the NS3 gene. The 3' part of this inserted sequence encodes anN-terminally truncated ubiquitin monomer. This is remarkable since alldescribed cp BVDV strains with ubiquitin coding sequences contain at leastone complete ubiquitin monomer. The 5' region of the nonviral sequence didnot show any homology to cellular sequences identified thus far in cp BVDVstrains. Databank searches revealed that this second cellular insertionencodes part of ribosomal protein S27a. Further analyses includedmolecular cloning and nucleotide sequencing of the cellular recombinationpartner. Sequence comparisons strongly suggest that the S27a and theubiquitin coding sequences found in the genome of CP Rit were both derivedfrom a bovine mRNA encoding a hybrid protein with the structureNH2-ubiquitin-S27a-COOH. Polyprotein processing in the genomic regionencoding the N-terminal part of NS4B, the two cellular insertions, and NS3was studied by a transient-expression assay. The respective analysesshowed that the S27a-derived polypeptide, together with the truncatedubiquitin, served as processing signal to yield NS3, whereas the truncatedubiquitin alone was not capable of mediating the cleavage. Since theexpression of NS3 is strictly correlated with the cp phenotype of BVDV,the altered genome organization leading to expression of NS3 most probablyrepresents the genetic basis of cytopathogenicity of CP Rit.

• Mezquita J, Pau M, Mezquita C
• Characterization and expression of two chicken cDNAs encoding ubiquitinfused to ribosomal proteins of 52 and 80 amino acids.
• Gene. 1997; 195: 313-9
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• We have determined the complete nucleotide sequence of two chicken cDNAs,Ub-t52 and Ub-t80, encoding ubiquitin fused to ribosomal proteins of 52and 80 amino acids. The deduced amino acid sequences of the ribosomalproteins are identical or very similar to the homologous human and ratproteins and to the corresponding proteins of other species. Unexpectedly,the ubiquitin moiety of the Ub-t52 protein showed two amino acidsubstitutions: serine-20 has been replaced by asparagine and serine-57 byalanine. Ubiquitin is a protein strongly conserved during evolution, withno changes in sequence previously reported in vertebrates. Ub-t52 andUb-t80 are highly expressed in early embryogenesis and during postmitoticstages of spermatogenesis, in parallel with the expression of thepolyubiquitin gene UbII. Whereas the 5' untranslated regions (5'UTRs) ofthe chicken polyubiquitin mRNAs showed marked differences in mature testesin relation to somatic tissues, no differences were observed in the 5'UTRsof the ubiquitin-ribosomal protein mRNAs. These mRNAs possess a5'-terminal oligopyrimidine tract that could be used as a mechanism topostpone translation during postmitotic stages of spermatogenesis, as hasbeen proposed in quiescent cells.

• Schreiber-Agus N, Torres R, Horner J, Lau A, Jamrich M, DePinho RA
• Comparative analysis of the expression and oncogenic activities of Xenopusc-, N-, and L-myc homologs.
• Mol Cell Biol. 1993; 13: 2456-68
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• A polymerase chain reaction-based cloning strategy allowed for theisolation of two distinct Xenopus L-myc genes, as well as previouslyisolated xc- and xN-myc genes, thus demonstrating that these threewell-defined members of the mammalian myc gene family are present in lowervertebrates as well. Comparison of the Xenopus and mammalian Myc familiesrevealed a high degree of structural relatedness at the gene and proteinlevels; this homology was consistent with the ability of the xc-myc1 andxN-myc1 genes to function as oncogenes in primary mammalian cells. Incontrast, the xL-myc1 gene was found to be incapable of transforming ratembryo fibroblast cells, and this inactivity may relate to localized butsignificant differences in its putative transactivation domain. Analysisof xc-, xN-, and xL-myc gene expression demonstrated that (i) all threegenes were highly expressed during oogenesis and their transcriptsaccumulated as abundant maternal mRNAs, (ii) each gene exhibited adistinctive pattern of expression during embryogenesis and in adulttissues, and (iii) the xL-myc1 and xL-myc2 genes were coordinatelyexpressed in the maternal and zygotic genomes. The markedly highexpression of the Xenopus myc gene family in differentiated tissues, suchas the central nervous system and kidney, contrasts sharply with the lowlevels observed in mammalian adult tissues. These differences may reflectunique functions of the Myc family proteins in processes specific toamphibians, such as tissue regeneration.