Showing papers on "Gene published in 1999"

Correlation between Protein and mRNA Abundance in Yeast

Abstract: The description of the state of a biological system by the quantitative measurement of the system constituents is an essential but largely unexplored area of biology. With recent technical advances including the development of differential display-PCR (21), of cDNA microarray and DNA chip technology (20, 27), and of serial analysis of gene expression (SAGE) (34, 35), it is now feasible to establish global and quantitative mRNA expression profiles of cells and tissues in species for which the sequence of all the genes is known. However, there is emerging evidence which suggests that mRNA expression patterns are necessary but are by themselves insufficient for the quantitative description of biological systems. This evidence includes discoveries of posttranscriptional mechanisms controlling the protein translation rate (15), the half-lives of specific proteins or mRNAs (33), and the intracellular location and molecular association of the protein products of expressed genes (32). Proteome analysis, defined as the analysis of the protein complement expressed by a genome (26), has been suggested as an approach to the quantitative description of the state of a biological system by the quantitative analysis of protein expression profiles (36). Proteome analysis is conceptually attractive because of its potential to determine properties of biological systems that are not apparent by DNA or mRNA sequence analysis alone. Such properties include the quantity of protein expression, the subcellular location, the state of modification, and the association with ligands, as well as the rate of change with time of such properties. In contrast to the genomes of a number of microorganisms (for a review, see reference 11) and the transcriptome of Saccharomyces cerevisiae (35), which have been entirely determined, no proteome map has been completed to date. The most common implementation of proteome analysis is the combination of two-dimensional gel electrophoresis (2DE) (isoelectric focusing-sodium dodecyl sulfate [SDS]-polyacrylamide gel electrophoresis) for the separation and quantitation of proteins with analytical methods for their identification. 2DE permits the separation, visualization, and quantitation of thousands of proteins reproducibly on a single gel (18, 24). By itself, 2DE is strictly a descriptive technique. The combination of 2DE with protein analytical techniques has added the possibility of establishing the identities of separated proteins (1, 2) and thus, in combination with quantitative mRNA analysis, of correlating quantitative protein and mRNA expression measurements of selected genes. The recent introduction of mass spectrometric protein analysis techniques has dramatically enhanced the throughput and sensitivity of protein identification to a level which now permits the large-scale analysis of proteins separated by 2DE. The techniques have reached a level of sensitivity that permits the identification of essentially any protein that is detectable in the gels by conventional protein staining (9, 29). Current protein analytical technology is based on the mass spectrometric generation of peptide fragment patterns that are idiotypic for the sequence of a protein. Protein identity is established by correlating such fragment patterns with sequence databases (10, 22, 37). Sophisticated computer software (8) has automated the entire process such that proteins are routinely identified with no human interpretation of peptide fragment patterns. In this study, we have analyzed the mRNA and protein levels of a group of genes expressed in exponentially growing cells of the yeast S. cerevisiae. Protein expression levels were quantified by metabolic labeling of the yeast proteins to a steady state, followed by 2DE and liquid scintillation counting of the selected, separated protein species. Separated proteins were identified by in-gel tryptic digestion of spots with subsequent analysis by microspray liquid chromatography-tandem mass spectrometry (LC-MS/MS) and sequence database searching. The corresponding mRNA transcript levels were calculated from SAGE frequency tables (35). This study, for the first time, explores a quantitative comparison of mRNA transcript and protein expression levels for a relatively large number of genes expressed in the same metabolic state. The resultant correlation is insufficient for prediction of protein levels from mRNA transcript levels. We have also compared the relative amounts of protein and mRNA with the respective codon bias values for the corresponding genes. This comparison indicates that codon bias by itself is insufficient to accurately predict either the mRNA or the protein expression levels of a gene. In addition, the results demonstrate that only highly expressed proteins are detectable by 2DE separation of total cell lysates and that therefore the construction of complete proteome maps with current technology will be very challenging, irrespective of the type of organism.

Animal mitochondrial genomes

Abstract: Animal mitochondrial DNA is a small, extrachromosomal genome, typically ~16 kb in size. With few exceptions, all animal mitochondrial genomes contain the same 37 genes: two for rRNAs, 13 for proteins and 22 for tRNAs. The products of these genes, along with RNAs and proteins imported from the cytoplasm, endow mitochondria with their own systems for DNA replication, transcription, mRNA processing and translation of proteins. The study of these genomes as they function in mitochondrial systems—‘mitochondrial genomics’— serves as a model for genome evolution. Furthermore, the comparison of animal mitochondrial gene arrangements has become a very powerful means for inferring ancient evolutionary relationships, since rearrangements appear to be unique, generally rare events that are unlikely to arise independently in separate evolutionary lineages. Complete mitochondrial gene arrangements have been published for 58 chordate species and 29 non-chordate species, and partial arrangements for hundreds of other taxa. This review compares and summarizes these gene arrangements and points out some of the questions that may be addressed by comparing mitochondrial systems.

Exploring the new world of the genome with DNA microarrays

Abstract: Thousands of genes are being discovered for the first time by sequencing the genomes of model organisms, an exhilarating reminder that much of the natural world remains to be explored at the molecular level. DNA microarrays provide a natural vehicle for this exploration. The model organisms are the first for which comprehensive genome-wide surveys of gene expression patterns or function are possible. The results can be viewed as maps that reflect the order and logic of the genetic program, rather than the physical order of genes on chromosomes. Exploration of the genome using DNA microarrays and other genome-scale technologies should narrow the gap in our knowledge of gene function and molecular biology between the currently-favoured model organisms and other species.

The transcriptional program in the response of human fibroblasts to serum.

Abstract: The temporal program of gene expression during a model physiological response of human cells, the response of fibroblasts to serum, was explored with a complementary DNA microarray representing about 8600 different human genes. Genes could be clustered into groups on the basis of their temporal patterns of expression in this program. Many features of the transcriptional program appeared to be related to the physiology of wound repair, suggesting that fibroblasts play a larger and richer role in this complex multicellular response than had previously been appreciated.

Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA

Abstract: Development of methods that allow an efficient expression of exogenous genes in animals would provide tools for gene function studies, treatment of diseases and for obtaining gene products. Therefore, we have developed a hydrodynamics-based procedure for expressing transgenes in mice by systemic administration of plasmid DNA. Using cDNA of luciferase and beta-galactosidase as a reporter gene, we demonstrated that an efficient gene transfer and expression can be achieved by a rapid injection of a large volume of DNA solution into animals via the tail vein. Among the organs expressing the transgene, the liver showed the highest level of gene expression. As high as 45 microg of luciferase protein per gram of liver can be achi- eved by a single tail vein injection of 5 microg of plasmid DNA into a mouse. Histochemical analysis using beta-galactosidase gene as a reporter reveals that approximately 40percent of hepatocytes express the transgene. The time-response curve shows that the level of transgene expression in the liver reaches the peak level in approximately 8 h after injection and decreases thereafter. The peak level of gene expression can be regained by repeated injection of plasmid DNA. These results suggest that a simple, convenient and efficient method has been developed and which can be used as an effective means for studying gene function, gene regulation and molecular pathophysiology through gene transfer, as well as for expressing proteins in animals.

Endotoxin-tolerant Mice Have Mutations in Toll-like Receptor 4 (Tlr4)

Abstract: Bacterial lipopolysaccharide (LPS) provokes a vigorous, generalized proinflammatory state in the infected host. Genetic regulation of this response has been localized to the Lps locus on mouse chromosome 4, through study of the C3H/HeJ and C57BL/10ScCr inbred strains. Both C3H/HeJ and C57BL/10ScCr mice are homozygous for a mutant Lps allele (Lpsd/d) that confers hyporesponsiveness to LPS challenge, and therefore exhibit natural tolerance to its lethal effects. Genetic and physical mapping of 1,345 backcross progeny segregating this mutant phenotype confined Lps to a 0.9-cM interval spanning 1.7 Mb. Three transcription units were identified within the candidate interval, including Toll-like receptor 4 (Tlr4), part of a protein family with members that have been implicated in LPS-induced cell signaling. C3H/HeJ mice have a point mutation within the coding region of the Tlr4 gene, resulting in a nonconservative substitution of a highly conserved proline by histidine at codon 712, whereas C57BL/ 10ScCr mice exhibit a deletion of Tlr4. Identification of distinct mutations involving the same gene at the Lps locus in two different hyporesponsive inbred mouse strains strongly supports the hypothesis that altered Tlr4 function is responsible for endotoxin tolerance.

c-Myc target genes involved in cell growth, apoptosis, and metabolism

Abstract: The c-myc gene was discovered as the cellular homolog of the retroviral v-myc oncogene 20 years ago (23, 25, 167). The c-myc proto-oncogene was subsequently found to be activated in various animal and human tumors (37, 39, 42). It belongs to the family of myc genes that includes B-myc, L-myc, N-myc, and s-myc; however, only c-myc, L-myc, and N-myc have neoplastic potential (54, 82, 102, 118, 178). Targeted homozygous deletion of the murine c-myc gene results in embryonic lethality, suggesting that it is critical for development (43). Homozygous inactivation of c-myc in rat fibroblasts caused a marked prolongation of cell doubling time, further suggesting a central role for c-myc in regulating cell proliferation (121). The frequency of genetic alterations of c-myc in human cancers (42) has allowed an estimation that approximately 70,000 U.S. cancer deaths per year are associated with changes in the c-myc gene or its expression. Given that c-myc may contribute to one-seventh of U.S. cancer deaths, recent efforts have been directed toward understanding the function of the c-Myc protein in cancer biology with the hope that therapeutic insights will emerge. Past efforts, which have contributed significantly to our current understanding of c-myc, are discussed in a number of excellent reviews (23, 29, 37, 40, 44, 52, 66, 82, 94, 102, 118, 125, 132, 145, 178, 182, 186).

Distinctive gene expression patterns in human mammary epithelial cells and breast cancers

Abstract: cDNA microarrays and a clustering algorithm were used to identify patterns of gene expression in human mammary epithelial cells growing in culture and in primary human breast tumors. Clusters of coexpressed genes identified through manipulations of mammary epithelial cells in vitro also showed consistent patterns of variation in expression among breast tumor samples. By using immunohistochemistry with antibodies against proteins encoded by a particular gene in a cluster, the identity of the cell type within the tumor specimen that contributed the observed gene expression pattern could be determined. Clusters of genes with coherent expression patterns in cultured cells and in the breast tumors samples could be related to specific features of biological variation among the samples. Two such clusters were found to have patterns that correlated with variation in cell proliferation rates and with activation of the IFN-regulated signal transduction pathway, respectively. Clusters of genes expressed by stromal cells and lymphocytes in the breast tumors also were identified in this analysis. These results support the feasibility and usefulness of this systematic approach to studying variation in gene expression patterns in human cancers as a means to dissect and classify solid tumors.

Evidence for lateral gene transfer between Archaea and Bacteria from genome sequence of Thermotoga maritima

Abstract: The 1,860,725-base-pair genome of Thermotoga maritima MSB8 contains 1,877 predicted coding regions, 1,014 (54%) of which have functional assignments and 863 (46%) of which are of unknown function. Genome analysis reveals numerous pathways involved in degradation of sugars and plant polysaccharides, and 108 genes that have orthologues only in the genomes of other thermophilic Eubacteria and Archaea. Of the Eubacteria sequenced to date, T. maritima has the highest percentage (24%) of genes that are most similar to archaeal genes. Eighty-one archaeal-like genes are clustered in 15 regions of the T. maritima genome that range in size from 4 to 20 kilobases. Conservation of gene order between T. maritima and Archaea in many of the clustered regions suggests that lateral gene transfer may have occurred between thermophilic Eubacteria and Archaea.

Targeted mRNA degradation by double-stranded RNA in vitro

Abstract: Double-stranded RNA (dsRNA) directs gene-specific, post-transcriptional silencing in many organisms, including vertebrates, and has provided a new tool for studying gene function. The biochemical mechanisms underlying this dsRNA interference (RNAi) are unknown. Here we report the development of a cell-free system from syncytial blastoderm Drosophila embryos that recapitulates many of the features of RNAi. The interference observed in this reaction is sequence specific, is promoted by dsRNA but not single-stranded RNA, functions by specific mRNA degradation, and requires a minimum length of dsRNA. Furthermore, preincubation of dsRNA potentiates its activity. These results demonstrate that RNAi can be mediated by sequence-specific processes in soluble reactions.

Patterns of single-nucleotide polymorphisms in candidate genes for blood-pressure homeostasis

Abstract: Sequence variation in human genes is largely confined to single-nucleotide polymorphisms (SNPs) and is valuable in tests of association with common diseases and pharmacogenetic traits. We performed a systematic and comprehensive survey of molecular variation to assess the nature, pattern and frequency of SNPs in 75 candidate human genes for blood-pressure homeostasis and hypertension. We assayed 28 Mb (190 kb in 148 alleles) of genomic sequence, comprising the 5´ and 3´ untranslated regions (UTRs), introns and coding sequence of these genes, for sequence differences in individuals of African and Northern European descent using high-density variant detection arrays (VDAs). We identified 874 candidate human SNPs, of which 22% were confirmed by DNA sequencing to reveal a discordancy rate of 21% for VDA detection. The SNPs detected have an average minor allele frequency of 11%, and 387 are within the coding sequence (cSNPs). Of all cSNPs, 54% lead to a predicted change in the protein sequence, implying a high level of human protein diversity. These protein-altering SNPs are 38% of the total number of such SNPs expected, are more likely to be population-specific and are rarer in the human population, directly demonstrating the effects of natural selection on human genes. Overall, the degree of nucleotide polymorphism across these human genes, and orthologous great ape sequences, is highly variable and is correlated with the effects of functional conservation on gene sequences.

Regulation of p53 in response to DNA damage

Abstract: Activation of p53 can occur in response to a number of cellular stresses, including DNA damage, hypoxia and nucleotide deprivation. Several forms of DNA damage have been shown to activate p53, including those generated by ionising radiation (IR), radio-mimetic drugs, ultraviolet light (UV) and chemicals such as methyl methane sulfonate (MMS). Under normal conditions, p53 levels are maintained at a low state by virtue of the extremely short-half life of the polypeptide. In addition to this, p53 normally exists in an largely inactive state that is relatively inefficient at binding to DNA and activating transcription. Activation of p53 in response to DNA damage is associated with a rapid increase in its levels and with an increased ability of p53 to bind DNA and mediate transcriptional activation. This then leads to the activation of a number of genes whose products trigger cell-cycle arrest, apoptosis, or DNA repair. Recent work has suggested that this regulation is brought about largely through DNA damage triggering a series of phosphorylation, de-phosphorylation and acetylation events on the p53 polypeptide. Here, we discuss the nature of these modifications, the enzymes that bring them about, and how changes in p53 modification lead to p53 activation.

Evolutionary relationships among diverse bacteriophages and prophages: All the world’s a phage

Abstract: We report DNA and predicted protein sequence similarities, implying homology, among genes of double-stranded DNA (dsDNA) bacteriophages and prophages spanning a broad phylogenetic range of host bacteria. The sequence matches reported here establish genetic connections, not always direct, among the lambdoid phages of Escherichia coli, phage phiC31 of Streptomyces, phages of Mycobacterium, a previously unrecognized cryptic prophage, phiflu, in the Haemophilus influenzae genome, and two small prophage-like elements, phiRv1 and phiRv2, in the genome of Mycobacterium tuberculosis. The results imply that these phage genes, and very possibly all of the dsDNA tailed phages, share common ancestry. We propose a model for the genetic structure and dynamics of the global phage population in which all dsDNA phage genomes are mosaics with access, by horizontal exchange, to a large common genetic pool but in which access to the gene pool is not uniform for all phage.

Heterozygous germ line hCHK2 mutations in Li-Fraumeni syndrome.

Abstract: The hCHK2 gene encodes the human homolog of the yeast Cds1 and Rad53 G2 checkpoint kinases, whose activation in response to DNA damage prevents cellular entry into mitosis. Here, it is shown that heterozygous germ line mutations in hCHK2 occur in Li-Fraumeni syndrome, a highly penetrant familial cancer phenotype usually associated with inherited mutations in the TP53 gene. These observations suggest that hCHK2 is a tumor suppressor gene conferring predisposition to sarcoma, breast cancer, and brain tumors, and they also provide a link between the central role of p53 inactivation in human cancer and the well-defined G2 checkpoint in yeast.

A Single Nucleotide Difference That Alters Splicing Patterns Distinguishes the SMA Gene SMN1 From the Copy Gene SMN2

Abstract: Spinal muscular atrophy (SMA) is a recessive disorder characterized by loss of motor neurons in the spinalcord. It is caused by mutations in the telomeric survival motor neuron 1 (SMN1) gene. Alterations within analmost identical copy gene, the centromeric survival motor neuron 2 (SMN2) gene produce no known pheno-typic effect. The exons of the two genes differ by just two nucleotides, neither of which alters the encodedamino acids. At the genomic level, only five nucleotides that differentiate the two genes from one anotherhave been reported. The entire genomic sequence of the two genes has not been determined. Thus, differ-ences which might explain why SMN1is the SMA gene are not readily apparent. In this study, we have com-pletely sequenced and compared genomic clones containing the SMNgenes. The two genes show strikingsimilarity, with the homology being unprecedented between two different yet functional genes. The only crit-ical difference in an ~32 kb region between the two SMNgenes is the C→→→→T base change 6 bp inside exon 7.This alteration but not other variations in the SMNgenes affects the splicing pattern of the genes. The majorityof the transcript from the SMN1locus is full length, whereas the majority of the transcript produced by theSMN2locus lacks exon 7. We suggest that the exon 7 nucleotide change affects the activity of an exon spliceenhancer. In SMA patients, the loss of SMN1but the presence of SMN2results in low levels of full-lengthSMNtranscript and therefore low SMN protein levels which causes SMA.INTRODUCTIONProximal spinal muscular atrophy (SMA) is an autosomalrecessive neuromuscular disorder characterized by destructionof motor neurons in the anterior horn of the spinal cord. SMAhas an estimated incidence of 1 in 10 000 live births, with a car-rier frequency of ~1 in 50 people (1). Childhood onset SMA isclassified into three groups on the basis of age at onset andclinical course (2); type I SMA (Werdnig–Hoffman disease) isthe most severe form, with onset before the age of 6 monthsand death usually occurring within the first 2 years. Type IISMA is intermediate in severity. Onset occurs at ~18 monthsand patients never gain the ability to walk. Type III SMA(Kugelberg–Welander disease) is the mildest form of the dis-ease with onset after 18 months. Type III patients are able tostand and walk.All three forms of proximal SMA are due to mutations in thetelomeric but not centromeric survival motor neuron (SMN)genes (3–11). The full-length cDNAs of the two genes areidentical except for single nucleotide differences in exons 7and 8, yet their transcriptional products are not the same.SMN1 produces a majority of the full-length cDNA;SMN2produces mostly transcript lacking exon 7 (3). We have shownpreviously that promoter differences do not account for the dif-ferent levels of full-length transcript from the two genes (12).Instead, the exon 7 difference between the two genes affectssplicing, causing increased levels of full-length transcript fromSMN1 as compared with SMN2 (13).The SMN protein is a 38 kDa polypeptide which is ubiqui-tously expressed (14,15). It is found at especially high levels inthe spinal motor neurons. The exact function of the proteinremains unknown. However, recent studies have implicated itsinvolvement in mRNA biogenesis. Specifically, SMN has been

Expression pattern and, surprisingly, gene length shape codon usage in Caenorhabditis, Drosophila, and Arabidopsis

Abstract: We measured the expression pattern and analyzed codon usage in 8,133, 1,550, and 2,917 genes, respectively, from Caenorhabditis elegans, Drosophila melanogaster, and Arabidopsis thaliana. In those three species, we observed a clear correlation between codon usage and gene expression levels and showed that this correlation is not due to a mutational bias. This provides direct evidence for selection on silent sites in those three distantly related multicellular eukaryotes. Surprisingly, there is a strong negative correlation between codon usage and protein length. This effect is not due to a smaller size of highly expressed proteins. Thus, for a same-expression pattern, the selective pressure on codon usage appears to be lower in genes encoding long rather than short proteins. This puzzling observation is not predicted by any of the current models of selection on codon usage and thus raises the question of how translation efficiency affects fitness in multicellular organisms.

The Berkeley Drosophila Genome Project gene disruption project: Single P-element insertions mutating 25% of vital Drosophila genes.

Abstract: A fundamental goal of genetics and functional genomics is to identify and mutate every gene in model organisms such as Drosophila melanogaster. The Berkeley Drosophila Genome Project (BDGP) gene disruption project generates single P-element insertion strains that each mutate unique genomic open reading frames. Such strains strongly facilitate further genetic and molecular studies of the disrupted loci, but it has remained unclear if P elements can be used to mutate all Drosophila genes. We now report that the primary collection has grown to contain 1045 strains that disrupt more than 25% of the estimated 3600 Drosophila genes that are essential for adult viability. Of these P insertions, 67% have been verified by genetic tests to cause the associated recessive mutant phenotypes, and the validity of most of the remaining lines is predicted on statistical grounds. Sequences flanking >920 insertions have been determined to exactly position them in the genome and to identify 376 potentially affected transcripts from collections of EST sequences. Strains in the BDGP collection are available from the Bloomington Stock Center and have already assisted the research community in characterizing >250 Drosophila genes. The likely identity of 131 additional genes in the collection is reported here. Our results show that Drosophila genes have a wide range of sensitivity to inactivation by P elements, and provide a rationale for greatly expanding the BDGP primary collection based entirely on insertion site sequencing. We predict that this approach can bring >85% of all Drosophila open reading frames under experimental control.

Molecular cloning of cDNAs which are highly overexpressed in mitoxantrone-resistant cells: demonstration of homology to ABC transport genes.

Abstract: Reports of multiple distinct mitoxantrone-resistant sublines without overexpression of P-glycoprotein or the multidrug-resistance associated protein have raised the possibility of the existence of another major transporter conferring drug resistance. In the present study, a cDNA library from mitoxantrone-resistant S1-M1-80 human colon carcinoma cells was screened by differential hybridization. Two cDNAs of different lengths were isolated and designated MXR1 and MXR2. Sequencing revealed a high degree of homology for the cDNAs with Expressed Sequence Tag sequences previously identified as belonging to an ATP binding cassette transporter. Homology to the Drosophila white gene and its homologues was found for the predicted amino acid sequence. Using either cDNA as a probe in a Northern analysis demonstrated high levels of expression in the S1-M1-80 cells and in the human breast cancer subline, MCF-7 AdVp3000. Levels were lower in earlier steps of selection, and in partial revertants. The gene is amplified 10-12-fold in the MCF-7 AdVp3000 cells, but not in the S1-M1-80 cells These studies are consistent with the identification of a new ATP binding cassette transporter, which is overexpressed in mitoxantrone-resistant cells.

Sequence and analysis of chromosome 2 of the plant Arabidopsis thaliana

Abstract: Arabidopsis thaliana (Arabidopsis) is unique among plant model organisms in having a small genome (130-140 Mb), excellent physical and genetic maps, and little repetitive DNA. Here we report the sequence of chromosome 2 from the Columbia ecotype in two gap-free assemblies (contigs) of 3.6 and 16 megabases (Mb). The latter represents the longest published stretch of uninterrupted DNA sequence assembled from any organism to date. Chromosome 2 represents 15% of the genome and encodes 4,037 genes, 49% of which have no predicted function. Roughly 250 tandem gene duplications were found in addition to large-scale duplications of about 0.5 and 4.5 Mb between chromosomes 2 and 1 and between chromosomes 2 and 4, respectively. Sequencing of nearly 2 Mb within the genetically defined centromere revealed a low density of recognizable genes, and a high density and diverse range of vestigial and presumably inactive mobile elements. More unexpected is what appears to be a recent insertion of a continuous stretch of 75% of the mitochondrial genome into chromosome 2.

Rapid Hypothesis Testing with Candida albicans through Gene Disruption with Short Homology Regions

Abstract: Candida albicans is an opportunistic fungal pathogen. It exists as a benign commensal organism in healthy individuals but causes infections in susceptible individuals, such as those with diminished immune function (14). Molecular genetic analysis of C. albicans has permitted evaluation of antifungal drug targets and elucidation of requirements for infection and pathogenesis (16). New C. albicans genes have been identified frequently through sequence homology to known genes or gene families. Gene discovery has been facilitated greatly by access to much of the C. albicans genomic sequence (11). Now, the rate-limiting step in analysis of gene function in this diploid organism is the creation of a homozygous disruption mutant. Gene disruption has been accomplished through successive transformations with insertion/deletion alleles that are constructed in vitro (2, 7, 12). These methods have thus far required isolation of substantial DNA segments, and yet new genes of interest are often identified through DNA sequences of 400 to 600 bp (3a). We report here a rapid method for disruption of C. albicans genes with PCR products that contain short regions of homology to the genome.

The gene encoding ribosomal protein S19 is mutated in Diamond-Blackfan anaemia

Abstract: Diamond-Blackfan anaemia (DBA) is a constitutional erythroblastopenia characterized by absent or decreased erythroid precursors. The disease, previously mapped to human chromosome 19q13, is frequently associated with a variety of malformations. To identify the gene involved in DBA, we cloned the chromosome 19q13 breakpoint in a patient with a reciprocal X;19 chromosome translocation. The breakpoint occurred in the gene encoding ribosomal protein S19. Furthermore, we identified mutations in RPS19 in 10 of 40 unrelated DBA patients, including nonsense, frameshift, splice site and missense mutations, as well as two intragenic deletions. These mutations are associated with clinical features that suggest a function for RPS19 in erythropoiesis and embryogenesis.

Plant disease resistance genes encode members of an ancient and diverse protein family within the nucleotide‐binding superfamily

Abstract: The nucleotide binding site (NBS) is a characteristic domain of many plant resistance gene products. An increasing number of NBS-encoding sequences are being identified through gene cloning, PCR amplification with degenerate primers, and genome sequencing projects. The NBS domain was analyzed from 14 known plant resistance genes and more than 400 homologs, representing 26 genera of monocotyledonous, dicotyle-donous and one coniferous species. Two distinct groups of diverse sequences were identified, indicating divergence during evolution and an ancient origin for these sequences. One group was comprised of sequences encoding an N-terminal domain with Toll/Interleukin-1 receptor homology (TIR), including the known resistance genes, N, M, L6, RPP1 and RPP5. Surprisingly, this group was entirely absent from monocot species in searches of both random genomic sequences and large collections of ESTs. A second group contained monocot and dicot sequences, including the known resistance genes, RPS2, RPM1, I2, Mi, Dm3, Pi-B, Xa1, RPP8, RPS5 and Prf. Amino acid signatures in the conserved motifs comprising the NBS domain clearly distinguished these two groups. The Arabidopsis genome is estimated to contain approximately 200 genes that encode related NBS motifs; TIR sequences were more abundant and outnumber non-TIR sequences threefold. The Arabidopsis NBS sequences currently in the databases are located in approximately 21 genomic clusters and 14 isolated loci. NBS-encoding sequences may be more prevalent in rice. The wide distribution of these sequences in the plant kingdom and their prevalence in the Arabidopsis and rice genomes indicate that they are ancient, diverse and common in plants. Sequence inferences suggest that these genes encode a novel class of nucleotide-binding proteins.

The daf-2 gene network for longevity regulates oxidative stress resistance and Mn-superoxide dismutase gene expression in Caenorhabditis elegans

Abstract: Longevity is regulated by the daf-2 gene network in Caenorhabditis elegans. Mutations in the daf-2 gene, which encodes a member of the insulin receptor family, confer the life extension (Age) phenotype and the constitutive dauer (a growth-arrested larval form specialized for dispersal) formation phenotype. The Age phenotype is mutually potentiated by two life extension mutations in the daf-2 gene and the clk-1 gene, a homologue of yeast CAT5/COQ7 known to regulate ubiquinone biosynthesis. In this study, we demonstrated that the daf-2 mutation also conferred an oxidative stress resistance (Oxr) phenotype, which was also enhanced by the clk-1 mutation. Similar to the Age phenotype, the Oxr phenotype was regulated by the genetic pathway of insulin-like signaling from daf-2 to the daf-16 gene, a homologue of the HNF-3/forkhead transcription factor. These findings led us to examine whether the insulin-like signaling pathway regulates the gene expression of antioxidant defense enzymes. We found that the mRNA le...