NORMAL GENOMIC

• Peterfy M, Phan J, Xu P, Reue K
• Lipodystrophy in the fld mouse results from mutation of a new gene encoding a nuclear protein, lipin.
• Nat Genet. 2001; 27: 121-4
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• Mice carrying mutations in the fatty liver dystrophy (fld) gene have features of human lipodystrophy, a genetically heterogeneous group of disorders characterized by loss of body fat, fatty liver, hypertriglyceridemia and insulin resistance. Through positional cloning, we have isolated the gene responsible and characterized two independent mutant alleles, fld and fld(2J). The gene (Lpin1) encodes a novel nuclear protein which we have named lipin. Consistent with the observed reduction of adipose tissue mass in fld and fld(2J)mice, wild-type Lpin1 mRNA is expressed at high levels in adipose tissue and is induced during differentiation of 3T3-L1 pre-adipocytes. Our results indicate that lipin is required for normal adipose tissue development, and provide a candidate gene for human lipodystrophy. Lipin defines a novel family of nuclear proteins containing at least three members in mammalian species, and homologs in distantly related organisms from human to yeast.

• Sanyal K, Ghosh SK, Sinha P
• The MCM16 gene of the yeast Saccharomyces cerevisiae is required for chromosome segregation.
• Mol Gen Genet. 1998; 260: 242-50
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• We have cloned and characterized the MCM16 gene required for the maintenance of minichromosomes in the yeast Saccharomyces cerevisiae. This gene corresponds to a 181-amino acid ORF, YPR046W, on chromosome XVI. Mutant cells carrying minichromosomes accumulate them in higher copy numbers than do wild-type cells. Intact dicentric plasmid could be recovered from the mutant, in contrast to the wild-type, in which the plasmid suffered frequent deletions. A wild-type centromere, CEN6, acts as a block to the transcription of a reporter gene, such as beta-galactosidase. This block was less effective in the mutant than in the wild-type strain, suggesting alterations in kinetochore assembly in the former. The mutant also showed increased sensitivity to the antimitotic drugs benomyl and thiabendazole. The mcm16 mutation caused a high rate of loss of chromosome III, without any significant increase in the recombination frequency. A strain carrying a deletion-disruption derivative of the MCM16 gene was viable and, when compared to the wild-type, did not show any significant changes in growth rate or cell morphology at 16, 23 and 37 degrees C. These properties show that MCM16 is required for an important but nonessential role that governs the kinetochore-microtubule mediated process of chromosome segregation.

• Bonnefoy N, Kermorgant M, Brivet-Chevillotte P, Dujardin G
• Cloning by functional complementation, and inactivation, of the Schizosaccharomyces pombe homologue of the Saccharomyces cerevisiae gene ABC1.
• Mol Gen Genet. 1996; 251: 204-10
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• The Saccharomyces cerevisiae gene ABC1 is required for the correct functioning of the bc1 complex of the mitochondrial respiratory chain. By functional complementation of a S. cerevisiae abc1(-) mutant, we have cloned a Schizosaccharomyces pombe cDNA, whose predicted product is 50% identical to the Abc1 protein. Significant homology is also observed with bacterial, nematode, and even human amino acid sequences of unknown function, suggesting that the Abc1 protein is conserved through evolution. The cloned cDNA corresponds to a single S. pombe gene abc1Sp, located on chromosome II, expression of which is not regulated by the carbon source. Inactivation of the abc1Sp gene by homologous gene replacement causes a respiratory deficiency which is efficiently rescued by the expression of the S. cerevisiae ABC1 gene. The inactivated strain shows a drastic decrease in the bc1 complex activity. a decrease in cytochrome aa3 and a slow growth phenotype. To our knowledge, this is the first example of the inactivation of a respiratory gene in S. pombe. Our results highlight the fact that S. pombe growth is highly dependent upon respiration, and that S. pombe could represent a valuable model for studying nucleo-mitochondrial interactions in higher eukaryotes.

• Couzin N, Trezeguet V, Le Saux A, Lauquin GJ
• Cloning of the gene encoding the mitochondrial adenine nucleotide carrier of Schizosaccharomyces pombe by functional complementation in Saccharomyces cerevisiae.
• Gene. 1996; 171: 113-7
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• We describe the isolation and sequencing of both cDNA and genomic clones encoding the mitochondrial ADP/ATP carrier (Anc) of Schizosaccharomyces pombe (Sp). The cDNA clone was isolated from a cDNA library of this fission yeast by complementation of a Saccharomyces cerevisiae (Sc) strain defective in adenine nucleotide carrier. The predicted amino acid (aa) sequence (322 aa) shared similarity with the known Anc sequences. It is more closely related to Neurospora crassa (Nc) Anc than to ScAnc1, 2, or 3 or Kluyveromyces lactis (Kl) Anc. Hybridization experiments with ordered libraries of Sp genomic DNA led to the physical mapping (chromosome II, NotI-B region) and the isolation of the Sp ANC1 gene. We also conclude that a single-copy gene encodes the Sp Anc.

• Wintersberger U, Kuhne C, Karwan A
• Scp160p, a new yeast protein associated with the nuclear membrane and the endoplasmic reticulum, is necessary for maintenance of exact ploidy.
• Yeast. 1995; 11: 929-44
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• We have cloned a new gene, SCP160, from Saccharomyces cerevisiae, the deduced amino acid sequence of which does not exhibit overall similarity to any known yeast protein. A weak resemblance between the C-terminal part of the Scp160 protein and regulatory subunits of cAMP-dependent protein kinases from eukaryotes as well as the pstB protein of Escherichia coli was observed. The SCP160 gene resides on the left arm of chromosome X and codes for a polypeptide of molecular weight around 160 kDa. By immunofluorescence microscopy the Scp160 protein appears to be localized to the nuclear envelope and to the endoplasmic reticulum (ER). However, no signal sequence or membrane-spanning region exists, suggesting that the Scp160 protein is attached to the cytoplasmic surface of the ER-nuclear envelope membranes. Disruption of the SCP160 gene is not lethal but results in cells of decreased viability, abnormal morphology and increased DNA content. This phenotype is not reversible by transformation with a plasmid carrying the wild-type gene. Crosses of SCP160 deletion mutant strains among each other or with unrelated strains lead to irregular segregation of genetic markers. Taken together the data suggest that the Scp160 protein is required during cell division for faithful partitioning of the ER-nuclear envelope membranes which in S. cerevisiae enclose the duplicated chromosomes.

• Belhumeur P, Lee A, Tam R, DiPaolo T, Fortin N, Clark MW
• GSP1 and GSP2, genetic suppressors of the prp20-1 mutant in Saccharomyces cerevisiae: GTP-binding proteins involved in the maintenance of nuclear organization.
• Mol Cell Biol. 1993; 13: 2152-61
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• The temperature-sensitive mutation prp20-1 of Saccharomyces cerevisiae exhibits a pleiotropic phenotype associated with a general failure to maintain a proper organization of the nucleus. Its mammalian homolog, RCC1, is not only reported to be involved in the negative control of chromosome condensation but is also believed to assist in the coupling of DNA replication to the entry into mitosis. Recent studies on Xenopus RCC1 have strongly suggested a further role for this protein in the formation or maintenance of the DNA replication machinery. To elucidate the nature of the various components required for this PRP20 control pathway in S. cerevisiae, we undertook a search for multicopy suppressors of a prp20 thermosensitive mutant. Two genes, GSP1 and GSP2, were identified that encode almost identical polypeptides of 219 and 220 amino acids. Sequence analyses of these proteins show them to contain the ras consensus domains involved in GTP binding and metabolism. The levels of the GSP1 transcript are about 10-fold those of GSP2. As for S. cerevisiae RAS2, GSP2 expression exhibits carbon source dependency, while GSP1 expression does not. GSP1 is an essential gene, and GSP2 is not required for cell viability. We show that GSP1p is nuclear, that it can bind GTP in an in vitro assay, and finally, that a mutation in GSP1p which activates small ras-like proteins by increasing the stability of the GTP-bound form causes a dominant lethal phenotype. We believe that these two gene products may serve in regulating the activities of the multicomponent PRP20 complex.