Novel actin-like proteins T-ACTIN 1 and T-ACTIN 2 are differentially expressed in the cytoplasm and nucleus of mouse haploid germ cells

We isolated cDNA clones for the novel actin-like proteins T-ACTIN 1 and T-ACTIN 2, which are expressed specifically in the mouse testis. These clones were from a subtracted cDNA library that was enriched for haploid germ cell-specific cDNAs. The mRNA sizes and deduced molecular masses of t-actin 1/mACTl7b and t-actin 2/mACTl7a were 2.2 kilobases (kb) and 1.8 kb, and Mr 43.1 x 10(3) and Mr 47.2 x 10(3), respectively. The two deduced amino acid sequences had 60% homology, and they had approximately 40% homology with other actins. The T-ACTINs contained some of the conserved regions seen in other actins. Although the cellular locations of these two proteins are quite different (T-ACTIN-1 was found in the cytoplasm and T-ACTIN-2 was located in the nucleus), the expression of their proteins and mRNAs is controlled during development and limited during spermiogenesis. In contrast, only T-ACTIN-2 was present in sperm heads and tails. These results suggest that T-ACTINs play important roles in sperm function and in the specific morphogenesis of spermatozoa during spermiogenesis.     

Characterization of a third human thyroid hormone receptor coexpressed with other thyroid hormone receptors in several tissues

A cDNA that encodes a third type of human thyroid hormone receptor (hTR alpha 1) has been isolated from a skeletal muscle library. The cDNA encodes a 410 amino acid protein, Mr = 46,820. When expressed and translated in vitro, hTR alpha 1 binds T3 with an association constant (ka) of 1.8 x 10(9) M-1. Comparison of the DNA sequence of hTR alpha 1 and a previously identified alpha type thyroid hormone receptor (hTR alpha 2) suggests that they could be transcribed from the same gene, and that alternative RNA splicing results in the synthesis of either hTR alpha 1 or hTR alpha 2. Two mRNA (3.2 kilobases and 6 kilobases) of hTR alpha 1 have been detected in several tissues. At least three types of thyroid hormone receptors (hTR alpha 1, alpha 2, beta), which possess similar affinities for hormone ligands, can be expressed in the same tissue.     

Organization of the human protein S genes

Human genomic clones that span the entire protein S expressed gene (PS alpha) and the 3' two-thirds of the protein S pseudogene (PS beta) have been isolated and characterized. The PS alpha gene is greater than 80 kilobases in length and contains 14 introns and 15 exons, as well as 6 repetitive "Alu" sequences. Exons I and XV contain 112 and 1139 bp 5' and 3' noncoding segments in addition to the amino and carboxyl termini, respectively. Exons I-VIII encode protein segments that are homologous to the vitamin K dependent clotting proteins and are bounded by introns whose position and type are identical with other members of this protein family. Exons IX-XV encode protein segments homologous to sex hormone binding globulin (SHBG) and are bounded by introns of identical type and position as in the SHBG gene. Genomic clones for the PS beta gene cover a distance of greater than 55 kilobases and contain segments corresponding to amino acids 46-635 of the mature protein and the 1.1-kb 3' noncoding region of the cDNA. The presence of multiple base changes in the coding portions of this gene, resulting in termination codons and frame shifts, suggests that it is a pseudogene. Comparison of DNA sequences for the two genes reveals 97% identity for coding and 3' noncoding, and 95.4% for intronic regions, suggesting divergence of the two genes is a relatively recent event.     

Characterization of a complementary deoxyribonucleic acid coding for human and bovine plasminogen

A cDNA library was constructed in pBR322 from bovine liver mRNA that was enriched for plasminogen mRNA by polysome immunoprecipitation. A 32P-labeled single-stranded cDNA was then prepared from the enriched bovine mRNA and employed as a probe to screen the cDNA library. The screening was carried out by testing for clones that protect the hybridized 32P-labeled cDNA from S1 nuclease digestion. The longest clone that was found was 581 base pairs in length and coded for the C-terminal 107 amino acids of bovine plasminogen, a 3' noncoding region of 246 nucleotides and a poly(A) tail. The bovine cDNA clone was then used as a probe to screen a human liver cDNA library of 18 000 recombinants. Six isolates were found to contain human plasminogen sequences. The longest clone consisted of 1851 base pairs corresponding to amino acid residues 272-790, followed by a 3' noncoding region of 227 base pairs and a poly(A) tail. Restriction fragments of the human cDNA were then used as probes to screen a human genomic DNA library present in a Charon 4A lambda phage library. Approximately 50 isolates from 10(6) recombinants were identified that hybridized to varying degrees with the cDNA probe. Among these, 10 corresponding to the gene for human plasminogen have been analyzed, and 3 that overlap have been shown to extend from kringle 3 through the 3' noncoding region of the gene. A 160 base pair exon with flanking splice junctions was then characterized and shown to encode for the first half of plasminogen kringle 4, including amino acid residues 346-399.     

Isolation and characterization of genomic clones for two chicken phenobarbital-inducible cytochrome P-450 genes

A cDNA clone specific for a chicken phenobarbital-inducible cytochrome P-450 was used to screen a chicken genomic library. Twenty-nine clones were isolated, restriction mapped, and divided into two non-overlapping groups. The cDNA clone hybridized to 12 kilobases of DNA from both groups. Both groups contained restriction fragments which hybridized to both 5' and 3' fragments of the cDNA clone, and it was concluded that the two groups were derived from two separate genes. Southern transfer analysis of individual chicken DNAs and quantitative hybridization analysis indicated that these two genes are independent and are present as single copies/haploid genome. Comparison of restriction digests of the cloned DNAs and total genomic DNA discounted the possibility that other closely related P-450 genes are present in the chicken genome.     

Molecular cloning and structural analysis of murine thymidine kinase genomic and cDNA sequences.

Two functional cytosolic thymidine kinase (tk) cDNA clones were isolated from a mouse L-cell library. An RNA blot analysis indicated that one of these clones contains a nearly full-length tk sequence and that LTK- cells contain little or no TK message. The nucleotide sequences of both clones were determined, and the functional mouse tk cDNA contains 1,156 base pairs. An analysis of the sequence implied that there is an untranslated 32-nucleotide region at the 5' end of the mRNA, followed by an open reading frame of 699 nucleotides. The 3' untranslated region is 422 nucleotides long. Thus, the gene codes for a protein containing 233 amino acids, with a molecular weight of 25,873. A comparison of the coding sequences of the mouse tk cDNA with the human and chicken tk genes revealed about 86 and 70% homology, respectively. We also isolated the tk gene from a mouse C57BL/10J cosmid library. The structural organization was determined by restriction mapping, Southern blotting, and heteroduplex analysis of the cloned sequences, in combination with a mouse tk cDNA. The tk gene spans approximately 11 kilobases and contains at least five introns. Southern blot analysis revealed that this gene is deleted in mouse LTK- cells, consistent with the inability of these cells to synthesize TK message. This analysis also showed that tk-related sequences are present in the genomes of several mouse strains, as well as in LTK- cells. These segments may represent pseudogenes.     

Nucleotide sequence of the gene for human prothrombin

A human genomic DNA library was screened for the gene coding for human prothrombin with a cDNA coding for the human protein. Eighty-one positive lambda phage were identified, and three were chosen for further characterization. These three phage hybridized with 5' and/or 3' probes prepared from the prothrombin cDNA. The complete DNA sequence of 21 kilobases of the human prothrombin gene was determined and included a 4.9-kilobase region that was previously sequenced. The gene for human prothrombin contains 14 exons separated by 13 intervening sequences. The exons range in size from 25 to 315 base pairs, while the introns range from 84 to 9447 base pairs. Ninety percent of the gene is composed of intervening sequence. All the intron splice junctions are consistent with sequences found in other eukaryotic genes, except for the presence of GC rather than GT on the 5' end of intervening sequence L. Thirty copies of Alu repetitive DNA and two copies of partial KpnI repeats were identified in clusters within several of the intervening sequences, and these repeats represent 40% of the DNA sequence of the gene. The size, distribution, and sequence homology of the introns within the gene were then compared to those of the genes for the other vitamin K dependent proteins and several other serine proteases.     

Human microsomal xenobiotic epoxide hydrolase. Complementary DNA sequence, complementary DNA-directed expression in COS-1 cells, and chromosomal localization

A lambda gt11 expression library constructed from human liver mRNA was screened with an antibody against human microsomal xenobiotic epoxide hydrolase. The clone pheh32 contains an insert of 1742 base pairs with an open reading frame coding for a protein of 455 amino acids with a calculated Mr of 52,956. The nucleotide sequence is 77% similar to the previously reported rat xenobiotic epoxide hydrolase cDNA sequence. The deduced amino acid sequence of the human epoxide hydrolase is 80% similar to the previously reported rabbit and 84% similar to the deduced rat protein sequence. The NH2-terminal amino acids deduced from the human xenobiotic epoxide hydrolase cDNA are identical to the published 19 NH2-terminal amino acids of the purified human xenobiotic epoxide hydrolase protein. Northern blot analysis revealed a single mRNA band of 1.8 kilobases. Southern blot analysis indicated that there is only one gene copy/haploid genome. The human xenobiotic epoxide hydrolase gene was assigned to the long arm of human chromosome 1. Several restriction fragment length polymorphisms were observed with the human epoxide hydrolase cDNA. pheh32 was expressed as enzymatically active protein in cultured monkey kidney cells (COS-1).     

A cDNA encoding protein kinase C identifies two species of mRNA in brain and GH3 cells

Antiserum raised against purified protein kinase C (the Ca2+/phospholipid-dependent enzyme) (Ballester, R., and rosen, O. M. (1985) J. Biol. Chem. 260, 15194-15199) was used to screen a rat brain cDNA library in the prokaryotic expression vector lambda gt11. Three positive clones were isolated and shown to have overlapping restriction endonuclease maps. The positive recombinant phage with the longest cDNA insert (1.4 kilobases (kb)) was used for production of a beta-galactosidase fusion protein. Rabbit antiserum raised against the fusion protein recognized a single rat brain polypeptide of Mr 80,000 which was identified as protein kinase C by the following criteria: electrophoretic co-migration with purified protein kinase C, partial co-purification with protein kinase C, and disappearance from the cytosol of phorbol 12-myristate 13-acetate-treated GH3 cells. The nick-translated cDNA hybridized with two mRNAs, 8 kb and 3.5 kb, whose tissue distribution was in agreement with that reported for protein kinase C activity. Hybrid selection with immobilized cDNA identified mRNA encoding a protein of Mr 80,000 that could be precipitated by antibody to purified protein kinase C. Treatment of GH3 cells with phorbol 12-myristate 13-acetate, which promotes translocation and subsequent degradation of protein kinase C, did not alter the level of either message.     

Structure of the gene for human von Willebrand factor

von Willebrand factor is a large multimeric plasma protein composed of identical subunits which contain four types of repeated domains. von Willebrand factor is essential for normal hemostasis, and deficiency of von Willebrand factor is the most common inherited bleeding disorder of man. Four human genomic DNA cosmid libraries and one bacteriophage lambda library were screened with von Willebrand factor cDNA probes. Twenty positive overlapping clones were characterized that span the entire von Willebrand factor gene. A high-resolution restriction map was constructed for approximately 75% of the locus and a total of approximately 33.8 kilobases was sequenced on both strands including all intron-exon boundaries. The gene is approximately 178 kilobases in length and contains 52 exons. The exons vary from 40 to 1379 base pairs in length, and the introns vary from 97 base pairs to approximately 19.9 kilobases in length. The signal peptide and propeptide (von Willebrand antigen II) of von Willebrand factor are encoded by 17 exons in approximately 80 kilobases of DNA while the mature subunit of von Willebrand factor and 3' noncoding region are encoded by 35 exons in the remaining approximately 100 kilobases of the gene. A number of repetitive sequences were identified including 14 Alu repeats and a approximately 670-base pair TCTA simple repeat in intron 40 that is polymorphic. Regions of the gene that encode homologous domains have similar structures, supporting a model for their origin by gene segment duplication.     

NAD(P)H:menadione oxidoreductase. Novel purification of enzyme cDNA and complete amino acid sequence, and gene regulation

NAD(P)H:menadione oxidoreductase induction by polycyclic hydrocarbons is known to be governed by the aromatic hydrocarbon-responsive (Ah) locus. This cytosolic enzyme was isolated from 3-methylcholanthrene-treated rat liver by a rapid two-step procedure with the use of affinity gel purification and fast-protein liquid chromatography. Polyclonal antiserum to menadione reductase was raised in rabbits. On Western (immuno) blot analysis, large increases in this hepatic menadione reductase protein (NMOR1) of 3-methylcholanthrene-treated C57BL/6N but not DBA/2N mice confirmed that induction of this enzyme by 3-methyl-cholanthrene is regulated by the Ah receptor. A cDNA expression library was constructed in lambda gt11 and screened with antiserum. Positive cDNA clones were plaque purified and further characterized by showing enhanced hybridization to 3-methylcholanthrene-induced poly(A+) RNA from rats; the longest cDNA clone (1,501 base pairs) has an open reading frame (bases 75-899) and a nucleotide sequence consistent with a new gene family. On Northern blot analysis, a single 3-methylcholanthrene-inducible rat liver mRNA (approximately 1.6 kilobases) hybridizes to the cDNA probe. On Southern blot analysis a total of 14-16 kilobases of rat genomic DNA fragments hybridize to the cDNA probe, indicating one or a small number of menadione reductase genes in this family. The amino acid sequence (274 residues) and Mr of 30,946 compare well with the size of the rat enzyme (32 kDa) estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amino acid sequence of two internal fragments of the trypsin-digested purified NMOR1 protein is in complete agreement with that deduced from the cDNA nucleotide sequence. This study represents the first cloning and sequencing of a cDNA encoding a Phase II drug-metabolizing enzyme under control of the Ah locus.