Human M2-type pyruvate kinase: cDNA cloning, chromosomal assignment and expression in hepatoma

Two overlapping clones, covering the entire coding sequence of human M2-type pyruvate kinase (PK) cDNA, were isolated and sequenced. Nucleotide sequencing results showed that they contained the 109-bp 5'-untranslated region, the 1593-bp coding region and the 585-bp 3'-untranslated region. Nucleotide sequence homology was 90% and 69% with rat M2-type and L-type PK cDNA, respectively. In situ hybridization using the human M2-type PK cDNA probe disclosed that the gene for M2-type PK is located at band q22 on chromosome 15. Northern blot analysis with RNA from human hepatoma demonstrated that M2-type PK was predominantly expressed in hepatoma cells, whereas L-type PK was preferentially expressed in the non-tumor portion of the liver.     

Characterization of two mRNA species encoding human estradiol 17 beta-dehydrogenase and assignment of the gene to chromosome 17

Using two 33-mer synthetic oligonucleotides derived from the amino acid sequence of the catalytic site of estradiol 17 beta-dehydrogenase (E2DH) and polyclonal antibodies raised against the enzyme purified from human placenta, clones were isolated from a lambda gt11 human placental cDNA library. A 327-amino acid sequence was deduced from cDNA sequencing. Two mRNA species have been identified in poly(A)+ RNA from human placenta, a major species migrating at 1.3 kb while a minor one is found at approx. 2.2 kb. Primer extension and S1 nuclease analysis indicate that the major mRNA species starts 9-10 nucleotides while the minor mRNA starts 971 nucleotides upstream from the ATG initiating codon, respectively. Sequence analysis of the longest cDNA clone (2092 bp) shows that it possesses identical coding and non-coding sequences in the regions of overlap with the shorter cDNA clones. The 32P-labeled 5' non-coding fragment hybridizes only to the 2.2 kb band, thus providing evidence for the existence of two distinct mRNA species which differ in their 5' noncoding regions. Using hp E2DH-36 cDNA as a probe for in situ hybridization of translocated chromosomes, the human E2DH gene was localized to the q11-q12 region of chromosome 17.     

Molecular cloning, cDNA structure and predicted amino acid sequence of bovine 3 beta-hydroxy-5-ene steroid dehydrogenase/delta 5-delta 4 isomerase

We have used our recently characterized human 3 beta-hydroxy-5-ene steroid dehydrogenase/delta 5-delta 4-isomerase (3 beta-HSD) cDNA as probe to isolate cDNAs encoding bovine 3 beta-HSD from a bovine ovary lambda gtll cDNA library. Nucleotide sequence analysis of two overlapping cDNA clones of 1362 bp and 1536 bp in length predicts a protein of 372 amino acids with a calculated molecular mass of 42,093 (excluding the first Met). The deduced amino acid sequence of bovine 3 beta-HSD displays 79% homology with human 3 beta-HSD while the nucleotide sequence of the coding region shares 82% interspecies similarity. Hybridization of cloned cDNAs to bovine ovary poly(A)+ RNA shows the presence of an approximately 1.7 kb mRNA species.     

Skeletal muscle actin mRNA. Characterization of the 3' untranslated region.

Plasmids p749, p106, and p150 contain cDNA inserts complementary to rat skeletal muscle actin mRNA. Nucleotide sequence analysis indicates the following sequence relationships: p749 specifies codons 171 to 360; p150 specifies codons 357 to 374 together with 120 nucleotides of the 3'-non-translated region; p106 specifies the last actin amino acid codon, the termination codon and the entire 3' non-translated region. Plasmid p749 hybridized with RNA extracted from rat skeletal muscle, cardiac muscle, smooth (stomach) muscle, and from brain. It also hybridizes well with RNA extracted from skeletal muscle and brain of dog and chick. Plasmid p106 hybridized specifically with rat striated muscles (skeletal and cardiac muscle) mRNA but not with mRNA from rat stomach and from rat brain. It also hybridized to RNA extracted from skeletal muscle of rabbit and dog but not from chick. Thermal stability of the hybrids and sensitivity to S1 digestion also indicated substantial divergence between the 3' untranslated end of rat and dog skeletal muscle actins. The investigation shows that the coding regions of actin genes are highly conserved, whereas the 3' non-coding regions diverged considerably during evolution. Probes constructed from the 3' non-coding regions of actin mRNAs can be used to identify the various actin mRNA and actin genes.     

Characterization of genomic and complementary DNA sequence of human C-reactive protein, and comparison with the complementary DNA sequence of serum amyloid P component

Complementary and genomic DNA clones corresponding to the human C-reactive protein (CRP) mRNA and structural gene have been analyzed and compared. Nucleotide sequencing of the coding regions of both cDNA and genomic DNA revealed an additional 19 amino acid peptide not described in the published CRP amino acid sequence. The CRP gene contains a single 278 base pair intron within the codon specifying the third residue of mature CRP. The intron contains a repetitive sequence (GT)15G(GT)3 which is similar to structures capable of adopting the Z-DNA form. A comparison of CRP coding and amino acid sequences with those of serum amyloid P component revealed striking overall homology which was not uniform: a region of limited conservation is bounded by two highly conserved regions.     

Chicken poly(ADP-ribose) synthetase: complete deduced amino acid sequence and comparison with mammalian enzyme sequences.

The complete nucleotide (nt) sequence of the cDNA encoding the chicken poly(ADP-ribose) synthetase has been determined. Positive clones overlapping the 5' region or the 3' region of the cDNA have been isolated from a lambda gt 10 hen oviduct cDNA library using two human cDNA probes. The missing middle portion has been obtained by the polymerase chain reaction procedure. A single 3033-nt open reading frame from start codon to stop codon encodes a sequence of 1011 amino acid residues. The alignment of this sequence with those from human and mouse reveals overall identities of 79% and 77%, respectively. However, an identity of about 82% is obtained in the DNA-binding domain within the two zinc fingers, and an even higher similarity (85-87%) is observed in the NAD-binding domain. The isolated clones consistently hybridize on chicken Northern blots to an mRNA species of about 4 kb, whereas they do not cross-hybridize with RNA blots of Drosophila melanogaster. Thus, it appears that, even if the functional properties of the enzyme are maintained, the cDNA identity will be much decreased in nonvertebrate organisms.     

Single base substitution between human intestinal and hepatic apolipoprotein B mRNA detected by ribonuclease cleavage analysis

The molecular mechanism of human intestinal apolipoprotein (apo) B-48 synthesis has been elucidated by a combination of sequencing of cloned complementary DNAs and RNase cleavage analysis of RNA heteroduplex. All intestinal cDNA clones contained a single C to T base substitution in the codon CAA encoding Gln2153 in apoB-100 cDNA, resulting in a translational stop. One of the our intestinal apoB cDNA clones was polyadenylated 106 bases downstream from the stop codon, possibly producing a 7-kb apoB message in the intestine. RNase cleavage analysis of the RNA heteroduplex between hepatic or intestinal RNA and apoB cDNA-directed anti-sense RNA showed that this single C to U substitution may occur in most of intestinal apoB mRNA. These results suggested that human apoB-48 is mostly produced by apoB mRNA with an in-frame stop codon in the intestine.     

Molecular cloning of human immune interferon cDNA and its expression in eukaryotic cells.

Starting with mRNA derived from Staphylococcal enterotoxin A induced human splenocytes, dsDNA was synthesized and inserted into unique BamHI site of the eukaryotic expression vector pSV529 (1). A recombinant plasmid containing human immune interferon (IFN-gamma) cDNA was identified by hybridization of plasmid inserted DNA bound onto nitrocellulose filters with mRNA derived from SEA-induced splenocytes, translation of the eluted RNA in Xenopus laevis oocytes and assaying for IFN activity. Plasmids containing the entire human IFN-gamma cDNA sequence were identified by colony hybridization and were sequenced. A unique coding region was identified which predicted a protein of 166 amino acids, the 20 N-terminal amino acids of which presumably represent a signal peptide. After transfection of monkey cells with plasmid DNA isolated from one of the recombinant clones (pHIIF-SV-gamma 1), IFN was excreted into the culture medium. This IFN was not distinguishable from human IFN-gamma by serological criteria or by cell target species specificity.     

Structure of mouse calpastatin isoforms: implications of species-common and species-specific alternative splicing.

Mouse calpastatin cDNAs were cloned by the method of RT-PCR using RNA isolated from myoblast C2C12 cells. Nucleotide sequencing of the isolated clones revealed an in-frame ATG codon upstream of the previously assigned translation initiation methionine. Except for the N-terminal segment, the new translatable region (domain XL) was similar to the sequence of bovine calpastatin in which domain XL was first identified. Among the isolated mouse calpastatin cDNA clones, three isoforms (mCS-a, mCS-b, and mCS-c) were identified. In domain L, mCS-b had a deletion of the region corresponding to exon 3 of the human calpastatin gene. RT-PCR analyses of various mouse tissues revealed that mCS-b was the major form and that the content of mCS-a, nondeleted form, was 5-10% in tissues including skeletal muscle, liver, brain, etc. and about 30% in the myoblast C2C12 cells. Unlike human and rat cDNAs, no other deletions were detected in mouse calpastatin domain L. Isolation of the cDNA clone of mCS-c, which lacked regions corresponding to exons 3 and 12, was obtained by chance because its expression level was under the detectable level in the mouse tissues and even in C2C12 cells.     

cDNA cloning and amino acid sequence for human myelin-associated glycoprotein

cDNA clones of human myelin-associated glycoprotein were isolated and analyzed. The combination of the two overlapping cDNA clones covered the full coding region and the complete amino acid sequence was deduced. In rat and mouse, expression of the two forms of mRNA is developmentally regulated; the mRNA without exon 12 portion is expressed mainly in the actively myelinating stage of development. Although the cDNA library used here was prepared from adult human brain poly(A)+ RNA, all five clones obtained corresponded to the mRNA without exon 12 portion.     

Human monocyte chemoattractant protein-1 (MCP-1). Full-length cDNA cloning, expression in mitogen-stimulated blood mononuclear leukocytes, and sequence similarity to mouse competence gene JE

The purpose of this work was to analyze cDNA encoding human monocyte chemoattractant protein-1 (MCP-1), previously isolated from glioma cell line culture fluid. Screening of a cDNA library from total poly(A) RNA of glioma cell line U-105MG yielded a clone that coded for the entire MCP-1. Nucleotide sequence analysis and comparison with the amino acid sequence of purified MCP-1 showed that the cDNA clone comprises a 53-nucleotide 5'-non-coding region, an open reading frame coding for a 99-residue protein of which the last 76 residues correspond exactly to pure MCP-1, and a 389-nucleotide 3'-untranslated region. The hydrophobicity of the first 23 residues is typical of a signal peptide. Southern blot analysis of human and animal genomic DNA showed that there is a single MCP-1 gene, which is conserved in several primates. MCP-1 mRNA was induced in human peripheral blood mononuclear leukocytes (PBMNLs) by PHA, LPS and IL-1, but not by IL-2, TNF, or IFN-gamma. Among proteins with similar sequences, the coding regions of MCP-1 and mouse JE show 68% identity. This suggest that MCP-1 is the human homologue of the mouse competence gene JE.     

Identification of a novel in-frame translational stop codon in human intestine apoB mRNA

Human apolipoprotein (apo) B exists in plasma as two isoproteins designated apoB-100 and apoB-48. ApoB-100 (512 kDa) and apoB-48 (250 kDa) are synthesized by the liver and intestine respectively. Analysis of apoB cDNA clones isolated from a human intestinal cDNA library revealed that the intestinal apoB mRNA contains a new in-frame translational stop codon. This premature stop codon is generated by a single base substitution of a 'C' to 'T' at nucleotide 6538 which converts the codon 'CAA' coding for the amino acid glutamine residue 2153 to an in-frame stop codon 'TAA'. The generation of a stop codon in the intestinal apoB mRNA appears to be tissue specific since it has not been reported in cDNA clones isolated from human liver cDNA libraries which code for the 4536 amino acid apoB-100. A potential polyadenylation signal sequence 'AATAAA' was also identified 390 bases downstream from the new stop codon. The new stop codon in the human intestinal apoB mRNA provides a potential mechanism for the biosynthesis of intestinal apoB-48.     

Use of the deoxyinosine-containing probe to isolate and sequence cDNA encoding the fusion (F) glycoprotein of Sendai virus (HVJ)

A synthetic 20-mer based on the known amino acid (aa) sequence of the N-terminus of Sendai virus F1 polypeptide was synthesized. Using this dI-probe, which contained deoxyinosines at all six ambiguous codon positions, we isolated clones carrying cDNAs for the F mRNA of Sendai virus. Nucleotide (nt) sequence analysis revealed a long open reading frame (ORF) that encodes a protein of 565 aa. Thus, this type of dI-probes should prove useful for selecting cDNA clones, when the aa sequence is known and is characterized by high codon redundancy.     

Cloning and characterization of the gene coding for cytoplasmic seryl-tRNA synthetase from Saccharomyces cerevisiae.

We have screened a Saccharomyces cerevisiae expression library with antibodies against seryl-tRNA synthetase (SerRS) from baker's yeast. In this way we obtained clones which contain serS, the structural gene for seryl-tRNA synthetase. Genomic Southern blots show that the serS gene resides on a 5.0 kb SalI fragment. Nucleotide sequence analysis of the genes revealed a single open reading frame from which we deduced the amino acid sequence of the enzyme consistent with that of two peptides isolated from SerRS. The enzyme is comprised of 462 amino acids consistent with earlier determinations of its molecular weight. The codon usage of serS is typical of abundant yeast proteins. Nuclease S1 analysis of serS mRNA defined the RNA initiation site 20-40 bases downstream from an AT rich sequence containing the TATA box and 21-39 nucleotides upstream of the translation initiation codon. Yeast strains transformed with the cloned gene overproduce seryl-tRNA synthetase in vivo.     

Cloning of the human oestrogen receptor cDNA

Poly A+ RNA isolated from the human breast cancer cell line MCF-7 was fractionated by sucrose gradient centrifugation and those fractions enriched in oestrogen receptor (ER) mRNA were used to prepare randomly primed cDNA libraries in the lambda gt11 vectors. Clones corresponding to the ER were isolated from both libraries after screening with either ER monoclonal antibodies (lambda gt11) or synthetic oligonucleotide probes designed from two peptide sequences of purified ER (lambda gt10). Five cDNA clones were isolated by antibody screening and five after screening with synthetic oligonucleotides. The two largest ER cDNA clones, lambda OR3 (1.3 kbase) and lambda OR8 (2.1 kbase), isolated using antibodies and oligonucleotides, respectively, were able to enrich selectively for ER mRNA by hybrid-selection. Furthermore, lambda OR8 contains DNA sequences which cross-hybridize with each of the other ER cDNA clones. These results demonstrate that the clones isolated correspond to the ER mRNA sequence. Using lambda OR8 as a hybridization probe revealed a single poly A+ RNA band of approx. 6.2 kbase in the ER containing human breast cancer cell lines MCF-7 and T47D. In contrast, no hybridization was seen in the human ER-cell line HeLa. The same probe hybridizes to a chicken gene which is expressed in oviduct tissue as a 7.5 kbase poly A+ RNA.     

Molecular cloning and sequence analysis of human Na,K-ATPase beta-subunit.

We have isolated a cDNA clone for the beta-subunit of HeLa cell Na,K-ATPase, containing a 2208-base-pair cDNA insert covering the whole coding region of the beta-subunit. Nucleotide sequence analysis revealed that the amino acid sequence of human Na,K-ATPase exhibited 61% homology with that of Torpedo counterpart (Noguchi et al. (1986) FEBS Lett. in press). A remarkable conservation in the nucleotide sequence of the 3' non-coding region was detected between the human and Torpedo cDNAs. RNA blot hybridization analysis revealed the presence of two mRNA species in HeLa cells. S1 nuclease mapping indicated that they were derived from utilization of two distinct polyadenylation signals in vivo. Total genomic Southern hybridization indicated the existence of only a few, possibly one set of gene encoding the Na,K-ATPase beta-subunit in the human genome.     

Molecular characterization of the murine interferon gamma receptor cDNA

Interferon gamma receptors (IFN-gamma R) exhibit remarkable species specificity. In order to understand the basis for this phenomenon, we have isolated a recombinant cDNA clone corresponding to the mouse (Mu) IFN-gamma R. Microinjection of the mRNA synthesized in vitro corresponding to the cloned cDNA into Xenopus laevis oocytes resulted in the synthesis of a protein that specifically binds Mu-IFN-gamma. Analysis of murine genomic and RNA blots with the cDNA probe indicates the presence of a single gene and a single mRNA species of about 2300 bases. Sequence analysis of the cDNA encoding the Mu-IFN-gamma R and comparison with the corresponding human IFN-gamma R sequence shows about 68% conservation of the extracellular domains and 51% conservation of the cytoplasmic domains at the nucleotide level. The results indicate that, as expected, the sequence of the receptor confers species specificity for the binding of IFN-gamma to the cell surface receptor. Moreover, it was previously shown that a human factor is required in addition to the receptor for the human IFN-gamma to function in hamster or mouse cells (Jung, V., Rashidbaigi, A., Jones, C., Tischfield, J.A., Shows, T.B., and Pestka, S. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 4151-4155). These results suggest an explanation for the second species-specific event required for function of the human receptor in mouse or hamster cells in that the intracellular domains are significantly different and thus cannot interact with the corresponding heterologous factor.