Amino acid sequence studies on lactate dehydrogenase C4 isozymes from mouse and rat testes

Carboxymethylated sperm-specific lactate dehydrogenase isozyme C4 (LDH-C4) proteins from mouse and rat testes were cleaved with cyanogen bromide and trypsin. Proteins were also citraconylated and digested with trypsin. In the case of mouse LDH-C4 isozyme, all 7 CNBr and 11 limited tryptic (arginine) peptides were isolated and sequenced. Some of the CNBr peptides were further fragmented with trypsin and chymotrypsin and their compositions and/or sequences characterized. Also, 34 of the 36 expected tryptic peptides were purified, and their compositions and sequences determined. Amino acid sequences of these peptides purified from mouse LDH-C4 were overlapped into a complete covalent structure of the 330 residues. For rat LDH-C4, 5 of 6 expected CNBr peptides, 5 of 8 expected arginine peptides, and 28 of the 34 expected tryptic peptides were isolated, and their compositions and sequences were determined. Some of the CNBr and arginine peptides were further fragmented with chymotrypsin, thermolysin, or V8 protease, and their compositions and/or sequences characterized. The amino acid sequence of 85% of the 330 residues from rat LDH-C subunit has been unambiguously determined, and the sequences of the remaining regions were tentatively aligned on the basis of peptide compositions and sequence homologies with the other known lactate dehydrogenase sequences, including mouse LDH-C. A comparison of the proposed rat LDH-C sequence with the complete covalent structure of mouse LDH-C indicates that 27 differences are located in the established rat LDH-C sequence of 280 residues and that 5 additional differences are in the tentative sequence of the remaining 50 amino acids.     

Characterization of the cDNA coding for mouse prothrombin and localization of the gene on mouse chromosome 2

A series of overlapping cDNAs coding for mouse prothrombin (coagulation factor II) have been isolated and the composite DNA sequence has been determined. The complete prothrombin cDNA is 1,987 bp in length [excluding the poly(A) tail] and codes for 18 bp of 5' untranslated sequence, an open reading frame coding for 618 amino acids, a stop codon, and a 3' untranslated region of 112 bp followed by a poly(A) tail. The translated amino acid sequence predicts a molecular weight of 66,087, which includes 10 residues of gamma-carboxyglutamic acid. There are five potential N-linked glycosylation sites. Mouse prothrombin is 81.4% and 77.3% identical to the human and bovine proteins, respectively. Comparison of the cDNA coding for mouse prothrombin to the human and bovine cDNAs indicates 79.9% and 76.5% identity, respectively. Amino acid residues important for the structure and function of human prothrombin are conserved in the mouse and bovine proteins. In the adult mouse and rat, prothrombin is primarily synthesized in the liver, where is constitutes 0.07% of total mRNA as determined by solution hybridization analysis. The genetic locus for mouse prothrombin, Cf-2, has been mapped using an interspecies backcross and DNA fragment differences between the two species. The prothrombin locus lies on mouse chromosome 2, 1.8 +/- 1.3 map units proximal to the catalase locus. The gene order in this region is Cen-Acra-Cf-2-Cas-1-A-Tel. This localization extends the proximal boundary of the known region of homology between mouse chromosome 2 and human chromosome 11p from Cas-1 about 2 map units toward the centromere.     

Sequence analysis of mouse cDNAs encoding ribosomal proteins L12 and L18

Mouse cDNAs encoding ribosomal proteins (r-proteins), L12 and L18, were isolated and their sequences determined. The L12 cDNA was found to contain 639 bp, including a coding sequence of 498 nucleotides (nt), 5' (78 nt) and 3' (45 nt) untranslated regions (UTRs), and a poly(A) tail of 18 nt. The L18 cDNA was shown to consist of 648 bp, including a coding sequence of 567 nt, 5' (26 nt) and 3' (39 nt) UTRs, and a poly(A) tail of 16 nt. The nt sequences of the protein-coding region from the mouse L12 and L18 cDNAs were found to exhibit 96% and 92% identity, respectively, with those of the rat. With the use of mouse L12 and L18 cDNA probes, multiple (at least 10) copies of the L12 and L18 gene families were shown to be present in the mouse and rat genomes. However, there was no sequence heterogeneity detected among seven L18 cDNA clones, indicating that only one copy of the L18 gene-related sequences is functional, and the other copies are presumably nonfunctional pseudogenes. The complete amino acid (aa) sequences of the mouse r-proteins, L12 and L18, were deduced from the nt sequences of their cDNA clones. L12 has 165 aa and a M_r, of 17 790, while L18 has 188 aa and a M_r of 21 570. The aa sequences of the mouse r-proteins, L12 and L18, exhibit 98% and 94% identity, respectively, to those of rat.     

Molecular cloning and sequence analysis of cDNA encoding human ferrochelatase

The cDNA encoding human ferrochelatase [EC 4.99.1.1] was isolated from a human placenta cDNA library in bacteriophage lambda gt11 by screening with a radiolabeled fragment of mouse ferrochelatase cDNA. The cDNA had an open reading frame of 1269 base pairs (bp) encoding a protein of 423 amino acid residues (Mr. 47,833) with alternative putative polyadenylation signals in the 3' non-coding regions and poly (A) tails. Amino acid sequencing showed that the mature protein consists of 369 amino acid residues (Mr. 42,158) with a putative leader sequence of 54 amino acid residues. The human enzyme showed an 88% identity to mouse enzyme and 46% to yeast enzyme. Northern blot analysis showed two mRNAs of about 2500 and 1600 bp for ferrochelatase in K562 and HepG2 cells. As full-length cDNA for human ferrochelatase is now available, molecular lesions related to erythropoietic protoporphyria can be characterized.     

Le projet „Génome Humain” et la caractérisation des étiquettes (E.S.T.) de testicule humain

The “Human Genome” is devoted to the identification of all the human genes and their chromosomal localization. Besides the sequencing of the genome, complementary strategies have been developed, including the characterization of mRNA by single pass sequencing of the corresponding cDNA. The partial sequences generated during this process represent expressed sequence tags (E.S.T.) of the corresponding genes. We used this strategy, associated with Northern blot andin situ hybridization, in order to characterize genes expressed in human testis. A cDNA library was prepared using mRNA purified from testes of a 27 years old man. We isolated, and stored in glycerol 7750 recombinant clones. 2200 clones with an insert over 700bp were single-pass sequenced, mainly at the 5' end, generating 2563 sequences products which were compared, with the sequences present in public databases. Out of 2000 sequences 481 were found identical to known human sequences; 358 were homologous to sequences from human or other species; 719 represented new sequences; 442 were rejected. Some of the clones corresponding to mRNAs transcribed from new genes were further analyzed on Northern blot of human, rat and mouse testes mRNA along with RNA of kidney, liver and brain. This allowed us to identify several new genes expressed in human testis of potential interest for the physiology of this tissue. Among others, they include a new mono-ADP ribosyl transferase and two testis specific poly A binding proteins.     

The baboon gene for apolipoprotein A-I: characterization of a cDNA clone and identification of DNA polymorphisms for genetic studies of cholesterol metabolism

We have isolated and sequenced a baboon apolipoprotein A-I (ApoA-I) clone from a liver cDNA library using a human cDNA hybridization probe. This baboon cDNA contains the entire ApoA-I coding region (801 bp, 267 aa), a 3' untranslated region, and a poly(A)tail. Among comparisons with apoAI sequences from other species, the baboon cDNA is most similar to that of the cynomolgus macaque (99.2% homologous) and least similar to the rat sequence (72.6% homologous). A high frequency of nonsynonymous substitutions are observed by alignment of baboon and human apoAI cDNAs, but comparisons of hydrophilicity profiles show that protein structure is conserved by substitutions of aa with similar properties. A polymorphic PstI cleavage site was identified by Southern blot analysis and subsequently mapped to the 5' end of the baboon apoAI gene. To identify effects of apoAI allelic variation on cholesterol metabolism, we used immunoblotting to compare the distributions of ApoA-I among lipoprotein size classes in baboons from each genotype under basal and atherogenic diets. We observed an increase of ApoA-I in high density lipoprotein (size class 1) particles after atherogenic diets in homozygotes for one allele, as compared to slight decreases in the other genotypes.     

A gene sequence expressed only in undifferentiated EC, EK cells and testes.

A clone, EC1, has been isolated from a cDNA library prepared from 4-day embryoid bodies formed by suspension culture of PSMB EC cells. This clone has been used to screen a variety of RNA sources including adult tissues, embryonal carcinoma (EC), and endoderm cell lines. A 3-kb poly(A)+ RNA species was found to be present only in undifferentiated EC cells and adult mouse testes. This species was significantly reduced in testes of W/Wv mice compared with wild-type at this locus. Germ cells and their progeny are therefore implicated as the source of the RNA in testes. Hybrid-selected RNA from PSMB could be translated in vitro into a 35-kd protein, but no translatable message was evident in either PYS-2 (parietal), or PSA5-E (visceral) endoderm cell lines. DNA sequencing of the EC1 insert revealed that it is 744 bp in length, the 3' 460 bp of which are in open reading frame. Comparison with known sequences have shown no significant homology. EC1 subclones in M13 have been used to generate single-stranded probes for hybridisation to RNA in situ in tissue sections. Hybridisation of the strand complementary to RNA produces a signal limited to the central regions of embryoid bodies formed on suspension culture of embryo-derived EK cells coinciding with the presence of undifferentiated cells. Probing of a mouse genomic library and Southern blots of liver DNA with EC1 reveals that the gene is present as a single copy.     

Molecular cloning of the alcohol/hydroxysteroid form (hSTa) of sulfotransferase from human liver.

A cDNA encoding the human alcohol/hydroxysteroid sulfotransferase (h-ST-a), which catalyzes the sulfo-conjugation of many drugs and hormones, was isolated from a human liver cDNA library using a rat STa (rSTa) cDNA probe. The cDNA, designated as hSTa, consists of 1069 base pairs (bp) and contains an 855-nucleotide open reading frame beginning at nucleotide 65, which encodes a 285 amino acid polypeptide of 33.76 kDa. A second cDNA clone (1563 bp) was truncated 5' at nucleotide 231 (lacking the first 15 amino acids) with identical coding region, however, it had a much longer 3' untranslated region (UTR). Both clones contained a short segment of poly(A)+ tail. Northern blot analysis of an adult human liver showed that there are at least 2 mature mRNA with sizes ranging from approximately 1.1 kb to 1.7 kb, verifying the authenticity of the obtained cDNA clones. From the sequence alignment, the hSTa shares 62%/74%, 39%/59%, 35%/48%, 36%/54% identity with rSTa, rSTp (phenol), rSTe (estrogen), and bovine STe (bSTe) at the deduced amino acid and DNA levels, respectively, indicating that there are at least three subfamilies (alcohol, phenol and estrogen) of genes that encode for sulfotransferases in mammals.     

Subfamilies of the mouse major urinary protein (MUP) multi-gene family: sequence analysis of cDNA clones and differential regulation in the liver

The mouse major urinary proteins (MUPs) are the products of a multi-gene family of 30-35 genes whose members exhibit diverse tissue specific, developmental, and hormonal controls. Three cDNA clones corresponding to liver MUP mRNAs have been sequenced. Two of the clones (p499, C57BL/6 and p1057, BALB/c) share strong homology whereas a third clone (p199, C57BL/6) has diverged considerably from the others at the nucleic acid (85% homology) and protein (68% homology) levels. The 5' regions of p499 and p199 which show the most sequence divergence were subcloned and shown to hybridize to different liver MUP mRNAs. The p499-5' sequence was expressed in all MUP expressing tissues (liver, lachrymal, submaxillary and mammary) whereas the p199-5' sequence was expressed primarily in the liver and lachrymal. Analysis of liver RNA from mice in different endocrine states indicates that the p499-5' sequence is strongly regulated by thyroxine administration whereas the p199-5' sequence is not. Both sequences appear to be regulated by growth hormone and testosterone. Southern blot analysis of mouse genomic DNA indicates that there are multiple genes homologous to each sequence.     

Linkage map of two HLA-SB beta and two HLA-SB alpha-related genes: an intron in one of the SB beta genes contains a processed pseudogene

Three overlapping cosmid clones contain coding sequences for four HLA Class II genes, provisionally identified as two HLA-SB alpha and two HLA-SB beta genes. The genes are in the order beta, alpha, beta, alpha, inverted with respect to each other. One of the SB beta genes contains a 513 bp sequence that appears to be a processed pseudogene, flanked by direct 17 bp repeat sequences, in the intron upstream of the beta 1 exon. The pseudogene is homologous to a family of sequences of approximately 25-40 members, most of which are not on chromosome 6. A cDNA clone, highly homologous to the pseudogene, except for its 5' end, contains a normal poly(A) addition site and a poly(A) tail. The cDNA clone is homologous to a single-copy gene in both man and mouse, encoded on human chromosome 15. A search of published DNA sequences identified a mouse sequence, with about 77% similarity to the pseudogene sequence, in the negative strand of an intron in a mouse dihydrofolate reductase gene. The second SB beta gene does not contain the pseudogene sequence.     

Identification of two cis-acting elements that independently regulate the length of poly(A) on Xenopus albumin pre-mRNA.

Unlike most eukaryotic mRNAs studied to date, Xenopus serum albumin mRNA has a short (17-residue), discrete poly(A) tail. We recently reported that this short poly(A) tail results from regulation of the length of poly(A) on albumin pre-mRNA. The purpose of the present study was to locate the cis-acting element responsible for this, the poly(A)-limiting element or PLE. An albumin minigene consisting of albumin cDNA joined in exon 13 to the 3' end of the albumin gene produced mRNA with <20 nt poly(A) when transfected into mouse fibroblasts. This result indicates both that cis-acting sequences that regulate poly(A) length are within this construct, and that nuclear regulation of poly(A) length is conserved between vertebrates. Poly(A) length regulation was retained after replacing the terminal 53 bp and 3' flanking region of the albumin gene with a synthetic polyadenylation element (SPA). Conversely, fusing albumin gene sequence spanning the terminal 53 bp of the albumin gene and 3' flanking sequence onto the human beta-globin gene yielded globin mRNA with a 200-residue poly(A)tail. These data indicate that the PLE resides upstream of the sequence elements involved in albumin pre-mRNA 3' processing. Poly(A) length regulation was restored upon fusing a segment bearing albumin intron 14, exon 15, and 3' flanking sequence onto the beta-globin gene. We demonstrate that exon 15 contains two PLEs that can act independently to regulate the length of poly(A).     

Complementary DNA and derived amino acid sequence of the alpha subunit of human complement protein C8: evidence for the existence of a separate alpha subunit messenger RNA

The entire amino acid sequence of the alpha subunit (Mr 64,000) of the eighth component of complement (C8) was determined by characterizing cDNA clones isolated from a human liver cDNA library. Two clones with overlapping inserts of net length 2.44 kilobases (kb) were isolated and found to contain the entire alpha coding region [1659 base pairs (bp)]. The 5' end consists of an untranslated region and a leader sequence of 30 amino acids. This sequence contains an apparent initiation Met, signal peptide, and propeptide which ends with an arginine-rich sequence that is characteristic of proteolytic processing sites found in the pro form of protein precursors. The 3' untranslated region contains two polyadenylation signals and a poly(A) sequence. RNA blot analysis of total cellular RNA from the human hepatoma cell line HepG2 revealed a message size of approximately 2.5 kb. Features of the 5' and 3' sequences and the message size suggest that a separate mRNA codes for alpha and argues against the occurrence of a single-chain precursor form of the disulfide-linked alpha-gamma subunit found in mature C8. Analysis of the derived amino acid sequence revealed several membrane surface seeking domains and a possible transmembrane domain. These occur in a cysteine-free region of the subunit and may constitute the structural basis for alpha interaction with target membranes. Analysis of the carbohydrate composition indicates 1 or 2 asparagine-linked but no O-linked oligosaccharide chains, a result consistent with predictions from the amino acid sequence.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and characterization of full-length mouse cDNA and genomic clones of 3-methylcholanthrene-inducible cytochrome P1-450 and P3-450

Polysome immuno-adsorption, with immunoglobulin G directed against two 3-methylcholanthrene-induced mouse liver cytochrome P-450 proteins, was used to enrich mRNA from 3-methylcholanthrene-treated C57BL/6N mouse liver. cDNA transcribed from the P-450-enriched mRNA was then cloned into the Okayama-Berg vector. Two cDNA classes were detected upon differential screening of the clone bank with [32P]cDNA derived from 3-methylcholanthrene-induced immuno-enriched versus control mRNA. Several representatives of these two classes were judged to be near full length by comparison with their corresponding mRNA mobilities on denaturing agarose gels. A continuous reading-frame near the 5' end of one cDNA class (P1-450) corresponds to a protein having 15 of 17 residues the same as the published N-terminal sequence of rat P-450c. A continuous reading frame near the 5' end of the other class (P3-450) corresponds exactly to the first 25 amino acids of the published N-terminal sequence of rat P-450d. The P1-450 cDNA is at least 700 bp longer than the P3-450 cDNA. Heteroduplex analysis and Southern blot hybridization demonstrate that these mRNAs share approx. 1100 bp of sequence homology. Genomic P1-450 and P3-450 clones were isolated from a gene library constructed from C57BL/6N mouse liver DNA. By heteroduplex analysis with the corresponding cDNA, the P1-450 gene spans about 6 kb and the P3-450 gene about 7 kb. The intron-exon patterns are very similar, with the second and seventh exons being much larger than the other five. The 3' terminal exon of P1-450 is about 500 bp longer than that of P3-450. These data suggest that both P1-450 and P3-450 have diverged from a common ancestral gene.