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1. A cDNA clone, named ppcMDH-1 and covering a part of the coding region for the porcine cytosolic malate dehydrogenase (cMDH) mRNA, was isolated from a porcine liver cDNA library. Subsequently, mouse cMDH cDNA clones were isolated from mouse liver and heart cDNA libraries, using the ppcMDH-1 cDNA as a probe. The longest clone, named pmcMDH-5, was sequenced and the primary structure of the mouse cMDH deduced from its cDNA sequence showed that the mouse cMDH consists of the 334-amino acid residues. When the amino acid sequence of the mouse cMDH was compared with that of the porcine cMDH, they shared a 93% homology. On the other hand, the amino acid sequences of mouse cMDH and mitochondrial MDH (mMDH) showed about 23% overall homology. Surprisingly, comparison of the amino acid sequences among the mammalian and bacterial MDHs revealed that the homology between the mouse cMDH and thermophilic bacterial MDH, as well as the homology between the mouse mMDH and Escherichia coli MDH, markedly exceeds the intraspecies sequence homology between mMDH and cMDH from mice. PMID: [3312200]
2. We cloned and characterized a mouse cytosolic malate dehydrogenase (cMDHase) (EC 1.1.1.37) gene, which is about 14 x 10(3) base-pairs long and is interrupted by eight introns. The 5' and 3' flanking regions and the exact sizes and boundaries of the exon blocks, including the transcription-initiation sites, were determined. The 5' end of the gene lacks the TATA and CAAT boxes characteristic of eukaryotic promoters, but contains G + C-rich sequences, one putative binding site for a cellular transcription factor, Sp1, and at least two major transcription-initiation sites. The sequences around the transcription-initiation sites are compatible with the formation of a number of potentially stable stem-loop structures. We compared structural organization of the mouse cMDHase gene with that of the previously characterized mouse mitochondrial MDHase (mMDHase) gene, and found that the conservation of intron positions spreads across much of the two genes. This result suggests that a common ancestral gene for the cytosolic MDHase and the mitochondrial MDHase was broken up by introns, before the divergence. We also compared the nucleotide sequence of the promoter region of the mouse cytosolic MDHase gene with that of the other three mouse genes coding for isoenzymes participating in the malate-aspartate shuttle, i.e. mitochondrial MDHase, cytosolic and mitochondrial aspartate aminotransferases (cAspATase and mAspATase). We found that highly conserved regions are present in the promoter region of the cAspATase gene. PMID: [3172222]
3. This study describes comprehensive polling of transcription start and termination sites and analysis of previously unidentified full-length complementary DNAs derived from the mouse genome. We identify the 5' and 3' boundaries of 181,047 transcripts with extensive variation in transcripts arising from alternative promoter usage, splicing, and polyadenylation. There are 16,247 new mouse protein-coding transcripts, including 5154 encoding previously unidentified proteins. Genomic mapping of the transcriptome reveals transcriptional forests, with overlapping transcription on both strands, separated by deserts in which few transcripts are observed. The data provide a comprehensive platform for the comparative analysis of mammalian transcriptional regulation in differentiation and development. PMID: [16141072]
4. The mouse (Mus musculus) is the premier animal model for understanding human disease and development. Here we show that a comprehensive understanding of mouse biology is only possible with the availability of a finished, high-quality genome assembly. The finished clone-based assembly of the mouse strain C57BL/6J reported here has over 175,000 fewer gaps and over 139 Mb more of novel sequence, compared with the earlier MGSCv3 draft genome assembly. In a comprehensive analysis of this revised genome sequence, we are now able to define 20,210 protein-coding genes, over a thousand more than predicted in the human genome (19,042 genes). In addition, we identified 439 long, non-protein-coding RNAs with evidence for transcribed orthologs in human. We analyzed the complex and repetitive landscape of 267 Mb of sequence that was missing or misassembled in the previously published assembly, and we provide insights into the reasons for its resistance to sequencing and assembly by whole-genome shotgun approaches. Duplicated regions within newly assembled sequence tend to be of more recent ancestry than duplicates in the published draft, correcting our initial understanding of recent evolution on the mouse lineage. These duplicates appear to be largely composed of sequence regions containing transposable elements and duplicated protein-coding genes; of these, some may be fixed in the mouse population, but at least 40% of segmentally duplicated sequences are copy number variable even among laboratory mouse strains. Mouse lineage-specific regions contain 3,767 genes drawn mainly from rapidly-changing gene families associated with reproductive functions. The finished mouse genome assembly, therefore, greatly improves our understanding of rodent-specific biology and allows the delineation of ancestral biological functions that are shared with human from derived functions that are not. PMID: [19468303]
5. The National Institutes of Health's Mammalian Gene Collection (MGC) project was designed to generate and sequence a publicly accessible cDNA resource containing a complete open reading frame (ORF) for every human and mouse gene. The project initially used a random strategy to select clones from a large number of cDNA libraries from diverse tissues. Candidate clones were chosen based on 5'-EST sequences, and then fully sequenced to high accuracy and analyzed by algorithms developed for this project. Currently, more than 11,000 human and 10,000 mouse genes are represented in MGC by at least one clone with a full ORF. The random selection approach is now reaching a saturation point, and a transition to protocols targeted at the missing transcripts is now required to complete the mouse and human collections. Comparison of the sequence of the MGC clones to reference genome sequences reveals that most cDNA clones are of very high sequence quality, although it is likely that some cDNAs may carry missense variants as a consequence of experimental artifact, such as PCR, cloning, or reverse transcriptase errors. Recently, a rat cDNA component was added to the project, and ongoing frog (Xenopus) and zebrafish (Danio) cDNA projects were expanded to take advantage of the high-throughput MGC pipeline. PMID: [15489334]
6. Though the interferon-inducible protein ISG15 was one of the first ubiquitin-like modifiers to be discovered, much remains unknown about the identity of proteins conjugated to ISG15 or the biologic consequences of modification. To gain a better understanding of the cellular pathways affected by ISG15, we identified proteins targeted for ISGylation using a proteomic approach. Mass spectrometric analysis identified 76 candidate ISGylation targets in anti-ISG15 immunoprecipitates from interferon-treated mouse or human cells. Twenty-one proteins were found in both mouse and human samples, including STAT1, a known target of ISGylation. Candidates identified in both species were tested for ISGylation in a transfection system: 18 of 19 proteins tested were ISGylated in this system. Two candidates, EF-2 and VCP, were also shown to be ISGylated in an interferon-dependent manner in the absence of exogenous over-expression. Seven proteins identified from a single species, but functionally related to candidates found in both species, were also ISGylated in the over-expression system. Proteins that can be ISGylated play important roles in translation, glycolysis, stress responses, and cell motility. These data indicate that ISGylation targets proteins found in several fundamentally important cellular pathways and will contribute to understanding the physiologic role of interferon-induced ISG15 and ISG15 conjugation. PMID: [16139798]
7. Metazoans employ reversible tyrosine phosphorylation to regulate innumerable biological processes. Thus, the large-scale identification of tyrosine phosphorylation sites from primary tissues is an essential step toward a molecular systems understanding of dynamic regulation in vivo. The relative paucity of phosphotyrosine has greatly limited its identification in large-scale phosphoproteomic experiments. However, using antiphosphotyrosine peptide immunoprecipitations, we report the largest study to date of tyrosine phosphorylation sites from primary tissue, identifying 414 unique tyrosine phosphorylation sites from murine brain. To measure the conservation of phosphorylated tyrosines and their surrounding residues, we constructed a computational pipeline and identified patterns of conservation within the signature of phosphotyrosine. PMID: [18034455]
8. Kinases play a prominent role in tumor development, pointing to the presence of specific phosphorylation patterns in tumor tissues. Here, we investigate whether recently developed high resolution mass spectrometric (MS) methods for proteome and phosphoproteome analysis can also be applied to solid tumors. As tumor model, we used TG3 mutant mice carrying skin melanomas. At total of 100 microg of solid tumor lysate yielded a melanoma proteome of 4443 identified proteins, including at least 88 putative melanoma markers previously found by cDNA microarray technology. Analysis of 2 mg of lysate from dissected melanoma with titansphere chromatography and 8 mg with strong cation exchange together resulted in the identification of more than 5600 phosphorylation sites on 2250 proteins. The phosphoproteome included many hits from pathways important in melanoma. One-month storage at -80 degrees C did not significantly decrease the number of identified phosphorylation sites. Thus, solid tumor can be analyzed by MS-based proteomics with similar efficiency as cell culture models and in amounts compatible with biopsies. PMID: [19367708]
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