Exon-intron organization and chromosomal localization of the mouse monoglyceride lipase gene

Monoglyceride lipase (MGL) functions together with hormone-sensitive lipase to hydrolyze intracellular triglyceride stores of adipocytes and other cells to fatty acids and glycerol. In addition, MGL presumably complements lipoprotein lipase in completing the hydrolysis of monoglycerides resulting from degradation of lipoprotein triglycerides. Cosmid clones containing the mouse MGL gene were isolated from a genomic library using the coding region of the mouse MGL cDNA as probe. Characterization of the clones obtained revealed that the mouse gene contains the coding sequence for MGL on seven exons, including a large terminal exon of approximately 2.6 kb containing the stop codon and the complete 3' untranslated region. Two different 5' leader sequences, diverging 21 bp upstream of the predicted translation initiation codon, were isolated from a mouse adipocyte cDNA library. Western blot analysis of different mouse tissues revealed protein size heterogeneities. The amino acid sequence derived from human MGL cDNA clones showed 84% identity with mouse MGL. The mouse MGL gene was mapped to chromosome 6 in a region with known homology to human chromosome 3q21.     

Human metallothionein genes: molecular cloning and sequence analysis of the mRNA.

From a cDNA clone bank prepared from cadmium-treated HeLa cells, we isolated clones representing mRNAs whose concentration is increased after cadmium induction. Several metallothionein cDNA clones were isolated by cross-hybridization to mouse metallothionein-I cDNA. The nucleotide sequence of one of these clones, containing a nearly full-length cDNA copy of human metallothionein-II mRNA, was determined. The homology between the human and mouse metallothionein sequences is strictly limited to the coding region of the mRNA. Codon usage in metallothionein mRNA is not random. Seventy-nine percent of the codons have G or C residues at the third position, resulting in a GC-rich sequence.     

Mouse cytoskeletal gamma-actin: analysis and implications of the structure of cloned cDNA and processed pseudogenes

The nucleotide sequence corresponding to almost the whole of a mouse gamma-cytoskeletal actin mRNA was determined from overlapping cloned DNA copies derived from brain mRNA. Several gamma-actin processed pseudogenes were isolated from a library of cloned DBA mouse genomic DNA, and the nucleotide sequences of these were determined and compared with that of the cDNA. This showed that two of these pseudogenes had arisen from a gene duplication or amplification event, and indicated that they had subsequently undergone partial correction against one another. The relative ages of the pseudogenes were estimated on the basis of their percentage divergence from the cDNA sequence and these were compared with an estimation based on the number of presumed silent mutations in the cDNA since each pseudogene had arisen. Consistent results were obtained, except in the case of one pseudogene which also showed an anomalous regional distribution of differences from the cDNA sequence. One way of accounting for the features of this anomalous pseudogene is by postulating that it is derived from a second functional gene for gamma-actin, different from that represented by the cDNA described here.     

Nucleotide sequence of cloned complementary deoxyribonucleic acid for the alpha subunit of bovine pituitary glycoprotein hormones

Recombinant DNA plasmids containing sequences coding for the alpha subunit of the bovine pituitary glycoprotein hormones have been isolated. The nucleotide sequences of three different cDNA clones have been determined. The largest alpha-subunit cDNA clone was found to contain 713 bases including 77 nucleotides from the 5'-untranslated region, 72 nucleotides coding for a precursor segment, 288 nucleotides coding for the mature alpha subunit, and 276 nucleotides from the 3'-untranslated region of the mRNA followed by a poly(A) segment. This cDNA likely represents most of the bovine alpha-subunit mRNA sequence. Nucleotide sequences were obtained from the cDNA inserts of two other alpha-subunit clones, and several differences among the three cDNA sequences have been detected. These differences in nucleotide sequence may represent either individual variation in genomic sequence or cloning artifacts. Comparison of the bovine alpha-subunit cDNA sequence to the sequences of human, rat, and mouse alpha-subunit cDNAs reveals that the bovine sequence has greater than 70% homology with the other cDNAs. The cloned alpha-subunit cDNA should provide a useful probe for further studies of the structure and expression of this interesting gene.     

Cloning and sequence analysis of cDNA for mouse prolactin

The present study was undertaken to find out whether or not sexual dimorphism in biological activities and amino acid compositions of mouse prolactin might be due to heterogeneity in mRNA for mouse prolactin Cloned cDNAs for mouse prolactin were first isolated from a mouse pituitary cDNA library by hybridization with a rat prolactin cDNA. Then, one clone of about 140 positive clones obtained from 2000 transformants was subjected to nucleotide sequence analysis and verified to contain a nearly full length of cDNA sequence coding for mouse prolactin precursor. The deduced complete amino acid sequence indicates that the precursor molecule consists of 31 amino acids as the signal peptide and 197 amino acids of prolactin, in which two amino acids were found to be different from the amino acid sequence previously published elsewhere. S1 nuclease mapping analysis using male and female pituitary RNAs indicates that mouse preprolactin is encoded by two mRNAs in both sexes. The two mRNAs differ from each other based upon the deletion of three nucleotides in the coding region for the signal peptide determined by the nucleotide sequence analysis in other cDNA clones. In the present study, no sexual difference was revealed in murine prolactin mRNA.     

Nucleotide sequence of mouse Tcp-1a cDNA

We have isolated complete cDNA clones encoding the mouse t-complex polypeptides 1A and 1B (TCP-1A and TCP-1B) from t-haplotype and wild-type (wt) mice, respectively. The complete nucleotide (nt) sequence of the Tcp-1a cDNA was determined. The Tcp-1a cDNA has an open reading frame (ORF) encoding a 60-kDa protein of 556 amino acids (aa). A comparison of nt sequences between the Tcp-1a and Tcp-1b cDNAs revealed that the 1786-bp regions upstream from their polyadenylation signals differed by 17 substitutions and that Tcp-1a had different polyadenylation sites from Tcp-1b. In these ORFs, 15 bp were substituted between the two alleles, occurring in 14 codons and resulting in eleven single-aa substitutions. Among these 15 substitutions, twelve were nonsynonymous (aa change) and three were synonymous (no aa change). The aa substitution in TCP-1 has occurred at least 20 times faster between t-haplotype and wt than between mouse and human or mouse and Drosophila.     

Phylogenetic conservation of a class III major histocompatibility complex antigen, factor B. Isolation and nucleotide sequencing of mouse factor B cDNA clones

The complement protein factor B is a novel serine protease which is encoded within the major histocompatibility complex in man, guinea pig, and mouse. To determine the structure of mouse factor B, cDNA clones were isolated from mouse strains of two different major histocompatibility complex haplotypes, H-2k and H-2d, and clones of 0.9 and 1.5 kilobases, respectively, were sequenced. The H-2d clone includes the H-2k clone sequence and spans 94% of the Bb-coding sequence. No differences in sequence or in restriction enzyme sites were observed between the H-2k and H-2d clones. The H-2d clone displays 83% nucleotide homology and 83% (derived) amino acid homology with that of human factor B; there are no insertions or deletions. Comparison of the mouse and human factor B sequence reveals extensive regional homology at the catalytic residues and in the NH2-terminal portion of the Bb fragment.     

Nucleotide sequence of mouse 5-aminolevulinic acid synthase cDNA and expression of its gene in hepatic and erythroid tissues

The cDNA coding for 5-aminolevulinic acid (ALA) synthase (EC 2.3.1.37) in both liver and anemic spleen of the mouse has been cloned. The liver clone was selected by complementation of an Escherichia coli hemA mutant. Erythroid clones were obtained by screening a cDNA library made from mouse anemic spleen RNA, using the liver cDNA as a probe. The sequences of the spleen-derived and liver-derived cDNAs are identical. The nucleotide sequence and predicted amino acid (aa) sequence of a 1.85-kb spleen-derived cDNA is presented. The mouse ALA synthase as sequence displays extensive homology to ALA synthase of chick embryonic liver. The ALA synthase mRNA, detected by Northern blot analysis, was the same size, approx. 2.3 kb, in mouse liver, anemic spleen, and mouse erythroleukemia cells. It is therefore unlikely that different isozymic forms of ALA synthase are present in mouse erythroid and hepatic tissue and this is not the basis for the different effects of heme and porphyrinogenic compounds on the expression of liver and erythroid ALA synthase.     

Cloning and sequencing of a cDNA encoding mouse stromelysin 1

A cDNA encoding mouse stromelysin 1 was cloned and the 1740-bp sequence was determined. The deduced amino acid (aa) sequence was compared with stromelysin 1 sequences of other mammals. Comparison with a previously published incomplete aa sequence of mouse stromelysin 1 revealed three single aa differences.     

Molecular isolation and sequence determination of the cDNA for the mouse sperm-specific lactate dehydrogenase-X gene

Several cDNA clones for the mouse lactate dehydrogenase-X (LDH-X), a sperm-specific glycolytic enzyme, were isolated from mouse testicular cDNA libraries constructed in the bacteriophage vectors, lambda gt11 and gt10. The largest cDNA clone contains an insert of 1135 base pairs in length and an open reading frame that encodes a 332 amino acid polypeptide with a molecular weight of 35.89 kD. The deduced amino acid sequence of this protein is in close agreement with the published sequence of mouse LDH-X obtained by direct protein sequencing. Northern analysis of RNA isolated from different tissues detected a single size mRNA of 1.5 kilobases in mouse testis but not in brain or liver. The Ldh-x structural gene was estimated to be about 12 kb in size as demonstrated by Southern hybridization analysis of mouse genomic DNA using the full-length cDNA as a probe.     

Drosophila RNA polymerase II elongation factor DmS-II has homology to mouse S-II and sequence similarity to yeast PPR2.

DmSII is a Drosophila RNA polymerase II elongation factor which suppresses pausing by RNA polymerase II at specific sites on double stranded templates. Using antibodies produced against the purified protein, a Drosophila cDNA expression library was screened and a cDNA was isolated which encoded a portion of DmSII. When this cDNA was used to probe Kc cell mRNA the predominant species was found to be 1.4 kb in length. The original cDNA was used to screen a Drosophila Kc cell cDNA library resulting in the isolation of a 1.4 kb cDNA which was then sequenced. The deduced protein sequence for DmSII exhibited high similarity to mouse SII protein sequence. In addition, significant sequence similarity was found with the protein encoded by the yeast gene PPR2, which is involved in regulation of URA4 gene expression. The comparison of amino acid sequences suggests that DmSII is comprised of two domains homologous to mouse SII separated by a flexible, serine rich region of low homology. The shorter yeast protein has sequence similarity only to the carboxy terminal domain.     

Cloning and sequencing of mouse GABA transporter complementary DNA

A cDNA encoding the mouse GABA transporter has been isolated and sequenced.The results show that the mouse GABA transporter cDNA differs from that of the rat by 60 base pairs at the open reading frame region but the deduced amino acid sequences of the two cDNAs are identical and both composed of 599 amino acids.However,the amino acid sequence is different from the sequence deduced from a recently published mouse GABA transporter cDNA.     

Cloning and expression of cDNA encoding mouse tyrosinase.

We have isolated a pigment cell-specific cDNA clone from a B16 mouse melanoma cDNA library by differential hybridization. The mRNA of isolated cDNA is highly expressed in B16 melanoma cells and in black mouse (C57BL/6) skin, but is not detectable in mouse neuroblastoma cells nor in K1735 mouse amelanotic melanoma cells. The protein sequence deduced from the nucleotide sequence of the cloned cDNA shows significant similarity to the entire region of Neurospora tyrosinase. To know the identity of cDNA, we transfected K1735 amelanotic melanoma and COS-7 cells with the cDNA carried in a simian virus 40 vector (pKCRH2). We confirmed that the isolated cDNA encodes mouse tyrosinase by immunofluorescence staining of transfected cells using two different anti-T4-tyrosinase monoclonal antibodies. Tyrosinase is composed of 513 amino acids with a molecular weight of 57,872 excluding a hydrophobic signal peptide of 24 amino acids.     

Mouse liver NAD(P)H:quinone acceptor oxidoreductase: protein sequence analysis by tandem mass spectrometry, cDNA cloning, expression in Escherichia coli, and enzyme activity analysis.

The amino acid sequence of mouse liver NAD(P)H:quinone acceptor oxidoreductase (EC 1.6.99.2) has been determined by tandem mass spectrometry and deduced from the nucleotide sequence of the cDNA encoding for the enzyme. The electrospray mass spectral analyses revealed, as previously reported (Prochaska HJ, Talalay P, 1986, J Biol Chem 261:1372-1378), that the 2 forms--the hydrophilic and hydrophobic forms--of the mouse liver quinone reductase have the same molecular weight. No amino acid sequence differences were found by tandem mass spectral analyses of tryptic peptides of the 2 forms. Moreover, the amino-termini of the mouse enzymes are acetylated as determined by tandem mass spectrometry. Further, only 1 cDNA species encoding for the quinone reductase was found. These results suggest that the 2 forms of the mouse quinone reductase have the same primary sequences, and that any difference between the 2 forms may be attributed to a labile posttranslational modification. Analysis of the mouse quinone reductase cDNA revealed that the enzyme is 273 amino acids long and has a sequence homologous to those of rat and human quinone reductases. In this study, the mouse quinone reductase cDNA was also ligated into a prokaryotic expression plasmid pKK233.2, and the constructed plasmid was used to transform Escherichia coli strain JM109. The E. coli-expressed mouse quinone reductase was purified and characterized. Although mouse quinone reductase has an amino acid sequence similar to those of the rat and human enzymes, the mouse enzyme has a higher NAD(P)H-menadione reductase activity and is less sensitive to flavones and dicoumarol, 2 known inhibitors of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prediction of the coding sequences of mouse homologues of KIAA gene: II. The complete nucleotide sequences of 400 mouse KIAA-homologous cDNAs identified by screening of terminal sequences of cDNA clones randomly sampled from size-fractionated libraries.

We have accumulated information of the coding sequences of uncharacterized human genes, which are known as KIAA genes, and the number of these genes exceeds 2000 at present. As an extension of this sequencing project, we recently have begun to accumulate mouse KIAA-homologous cDNAs, because it would be useful to prepare a set of human and mouse homologous cDNA pairs for further functional analysis of the KIAA genes. We herein present the entire sequences of 400 mouse KIAA cDNA clones and 4 novel cDNA clones which were incidentally identified during this project. Most of clones entirely sequenced in this study were selected by computer-assisted analysis of terminal sequences of the cDNAs. The average size of the 404 cDNA sequences reached 5.3 kb and that of the deduced amino acid sequences from these cDNAs was 868 amino acid residues. The results of sequence analyses of these clones showed that single mouse KIAA cDNAs bridged two different human KIAA cDNAs in some cases, which indicated that these two human KIAA cDNAs were derived from single genes although they had been supposed to originate from different genes. Furthermore, we successfully mapped all the mouse KIAA cDNAs along the genome using a recently published mouse genome draft sequence.     

The cDNA sequence of mouse LAMP-2. Evidence for two classes of lysosomal membrane glycoproteins

We describe the isolation and sequencing of a cDNA encoding the mouse lysosomal membrane glycoprotein mLAMP-2 and the sequence differences that distinguish this molecule from the LAMP-1 class of proteins. An oligonucleotide probe corresponding to the NH2-terminal amino acid sequence of purified mLAMP-2 was synthesized by the polymerase chain reaction and used to screen several cDNA libraries. cDNA clones with an insert of 1,700 nucleotides were identified and sequenced. The deduced amino acid sequence of mLAMP-2 comprises a signal sequence of 25 residues and a 390-amino acid polypeptide (Mr 43,017) with the following putative domains: a large intraluminal region (residues 1-354) with 17 N-linked glycosylation sites (Asn-X-Ser/Thr), a hydrophobic transmembrane-spanning region of 24 residues (355-378), and a COOH-terminal cytoplasmic tail of 12 residues (379-390). When this sequence is compared with those of other lysosomal membrane glycoproteins, it is apparent that mouse LAMP-2 and human LAMP-2 form one homology class (LAMP-2) that is separated from the LAMP-1 class of proteins. The sequence differences in these two classes provide a basis for comparing the structure of the proteins with their biochemical and biological properties.