Purification and characterization of human plasma Zn-alpha 2-glycoprotein

Zn-alpha 2-glycoprotein (Zn alpha 2gp) was purified from fresh human plasma approximately 670-fold in a yield of 18% over the fractions from DEAE-Sephadex A-50 column chromatography. The purified protein was a glycoprotein with molecular weights of 56,000 and 57,000 on Superose and Sephadex G-150 column chromatographies and of 41,000 and 42,000 on nonreduced SDS-PAGE. Characterization, which included a determination of molecular weight, amino acid composition, amino terminus, and antigenicity, correlated well with known values previously reported for human Zn alpha 2gp.     

Immunohistochemical localization of Zn-alpha 2-glycoprotein in normal human tissues

The Zn-alpha 2-glycoprotein (Zn-alpha 2-GP) is present at a high concentration in the seminal plasma and at significant levels in other human body fluids. Its precise localization, however, has remained unclear, as well as its physiological and pathological significance. The present study reports the immunohistochemical localization of this protein in normal adult human tissues. Localization of the reactive product to anti-human plasma Zn-alpha 2-GP antibody was demonstrated in the following cells: luminal and basal cells of the prostate gland, luminal epithelial cells of the acini and of some ducts of the mammary glands, luminal cells of the secretory portion of the eccrine and apocrine sweat glands, serous cells of the salivary, tracheal, and bronchial glands, acinar cells of the esophageal glands, exocrine acinar cells of the pancreas, hepatocytes of the liver, and epithelial cells of the proximal and distal tubules in the kidney. The present results suggest that Zn-alpha 2-GP exerts some unknown but fairly widespread exocrine function and may be produced in the various epithelial cells tested. Hepatocytes are also suggested to be a source of the protein in the blood plasma.     

Cloning and nucleotide sequence of cDNA encoding human liver serine-pyruvate aminotransferase

Cloned cDNAs for human liver serine-pyruvate aminotransferase (Ser-PyrAT) were obtained by screening of a human liver cDNA library with a fragment of cDNA for rat mitochondrial Ser-PyrAT as a probe. Two clones were isolated from 50,000 transformants. Both clones contained approximately 1.5 kb cDNA inserts and were shown to almost completely overlap each other on restriction enzyme mapping and DNA sequencing. The nucleotide sequence of the mRNA coding for human liver Ser-PyrAT was determined from those of the cDNA clones. The mRNA comprises at least 1487 nucleotides, and encodes a polypeptide consisting of 392 amino acid residues with a molecular mass of 43,039 Da. The amino acid composition determined on acid hydrolysis of the purified enzyme showed good agreement with that deduced from the nucleotide sequence of the cDNA. In vitro translation of the mRNA derived from one of the isolated clones, pHspt12, as well as that of mRNA extracted from human liver, yielded a product of 43 kDa which reacted with rabbit anti-(rat mitochondrial Ser-PyrAT) serum. Comparison of the deduced amino acid sequences of human Ser-PyrAT and the mature form of rat mitochondrial Ser-PyrAT revealed 79.3% identity. Although human Ser-PyrAT appears to be synthesized as the mature size, the 5'-noncoding region of human Ser-PyrAT mRNA contains a nucleotide sequence which would encode, if translated, an amino acid sequence similar to that of the N-terminal extension peptide of the precursor for rat mitochondrial Ser-PyrAT.     

Complete nucleotide sequence of the chromosomal gene for human IL-4 and its expression

We have isolated a chromosomal DNA segment of the human IL-4 gene based on homology with a human IL-4 cDNA sequence and determined its complete nucleotide sequence. The human IL-4 gene, which occurs as a single copy in the haploid genome, is mapped on chromosome 5. It is composed of four exons and three introns and is approximately 10 kilobase pairs in size. 5'-Flanking regions of human and mouse IL-4 genes share about 85% homology extending more than 500 base pairs upstream of a "TATA" like sequence. Several patches of sequences are found in the 5'-flanking region of the human IL-4 gene which are homologous to sequence in the 5'-flanking regions of the IL-2, IL-3, IL-5, and granulocyte-macrophage (GM)-CSF genes. The IL-4 gene is inducible after treatment of human T cell clone by phorbol-12-myristate-13-acetate (TPA) and calcium ionophore A23187. The 2.3-kb 5'-flanking region of the human IL-4 gene transiently transfected into Jurkat human T cell leukemia cells is activated efficiently in response to TPA and A23187 stimulation and, although less efficiently, by human T cell leukemia virus type I-encoded p40x or BPV-encoded E2 protein. Combination of TPA/A23187 and p40x or E2 protein further augmented the level of expression.     

Canine 5S rRNA: nucleotide sequence and chromosomal assignment of its gene cluster in four canid species

The purpose of this study was to determine the nucleotide sequence of canine 5S rRNA and use this information to develop a molecular probe to assign the gene locus to chromosomes of the dog and three other related canid species using fluorescence in situ hybridization. The nucleotide sequence of canine liver 5S rRNA is 120 base pairs long and identical to the 5S rRNA nucleotide sequence of all other mammalian species investigated so far. A single 5S rRNA gene cluster was localized pericentromerically on chromosomes of four canid species: dog 4q1.3, red fox 4q1.3, blue fox 3q1.3 and Chinese raccoon dog 8q1.3. Chromosome arms carrying the 5S rRNA gene cluster showed striking similarities in their QFQ banding patterns, suggesting high conservation of these chromosome arms among the four species studied. The chromosomal assignments of 5S rRNA genes are among the first gene mapping results for the blue fox and the Chinese raccoon dog, and are in accordance with published data on comparative chromosome maps from human, dog, red fox, blue fox and raccoon dogs.     

Cloning and nucleotide sequence of placental hGH-V cDNA

We have previously demonstrated the presence in human placenta and maternal serum of a GH variant, called human placental growth hormone (hPGH). We have also shown that the hGH-V gene is expressed at the placental level thus possibly coding for hPGH. The hGH-V cDNA has now been isolated from a lambda gt 11 human placenta cDNA library. Its sequence has been determined which firmly establishes the GH-V gene mode of splicing as well as the GH-V protein structure. Our data give final evidence of placental hGH-V gene expression and reinforce the hypothesis of identity between the hGH-V protein and hPGH.     

Cloning and nucleotide sequence of ovine prolactin cDNA

A cDNA expression library was constructed in the lambda gt 11 phage vector using ovine (o) pituitary mRNA. The clone, pOP1, carrying a 934-bp insert contains an open reading frame beginning with the first nucleotide (nt) and ending with the stop codon TAA at nt position 781. Two potential translation start codons (ATGs) are present in the 5' region of this cDNA. Translation initiation could occur at the 5' proximal ATG at nt position 61. The nucleotide sequence around this ATG (TCCATGG), resembles the optimum sequence context for translation initiation by the eukaryotic ribosomes, as defined by mutational analysis [Kozak, Cell 44 (1986) 283-292)], with its substitution of the A at -3 of the consensus sequence by a T residue in this clone. Translation initiated at this codon could potentially code for the entire pre-prolactin (pre-PRL) molecule. The 3'-untranslated region is 154 nt long and contains a polyadenylation signal AATAAA. The deduced amino acid sequence agrees in totality with the published amino acid sequence of the mature hormone. The present study reports on the nucleotide sequence of o-PRL mRNA and the deduced amino acid sequence in the signal peptide of the hormone.     

Cloning and nucleotide sequence of the murine hsp84 cDNA and chromosome assignment of related sequences

The nucleotide (nt) sequence of mouse 84-kDa heat shock protein (Hsp) cDNA has been determined using a combination of molecular cloning and oligodeoxynucleotide priming on poly(A) + RNA. The cDNA was 2.5 kb long, not including the poly(A) tail. It contained a 5' leader of about 94 nt that was G + C-rich, and a 243-nt 3'-untranslated region that was A + T-rich in the vicinity of the polyadenylation signal. Gene hsp84 codes for an acidic polypeptide of 724 amino acid (aa) residues. Mouse Hsp84 had 81% and 63% aa homology to Drosophila melanogaster Hsp82 and yeast Hsp90, respectively. The nucleotide sequence had 74% and 59% homology to Drosophila and yeast hsp sequences, respectively, in the coding regions of these genes. This homology did not extend to the 5' - and 3'-untranslated regions. Chromosomal analysis indicated that hsp84-related sequences are on at least three different chromosomes.     

Cloning and nucleotide sequence of a full-length cDNA for human 14 kDa beta-galactoside-binding lectin

A full-length cDNA for a 14K-type human lung beta-galactoside-binding lectin was cloned. The cDNA includes a 405 bp open reading frame coding 135 amino acids including the initiator methionine, and having a single internal EcoRI site and a polyadenylation signal. The deduced amino-acid sequence agreed completely with the sequence of a human placenta lectin determined by direct amino-acid sequence analysis (Hirabayashi, J. and Kasai, K. (1988) J. Biochem. 104, 1-4). It showed extensive sequence similarity with other vertebrate 14K-type lectins and a 35K-type lectin (carbohydrate-binding protein 35) of mouse 3T3 cell. Search of a Genbank sequence data base revealed significant sequence similarity between the beta-galactoside-binding lectins and the carboxyl-terminal half of an IgE-binding protein, the cDNA of which has been cloned from rat basophilic leukemia cells. Thus, 14K-type lectin, 35K-type lectin and IgE-binding protein appeared to form a superfamily of proteins. Almost all invariant residues are located in the central region of the 14K-type lectins, so this region may constitute an essential part of the lectins, such as the sugar-binding domain.     

Cloning and nucleotide sequence of a full-length cDNA for human liver gamma-glutamylcysteine synthetase.

We have cloned and sequenced a full-length cDNA for human liver gamma-glutamylcysteine synthetase (GCS), the rate-limiting enzyme in glutathione biosynthesis. The cDNA consists of 2634 bp containing an open reading frame encoding a protein of 367 amino acids and having a calculated M(r) = 72,773. The nucleotide sequence of the cDNA for human liver GCS shares an 84% overall similarity with the composite rat GCS sequence deduced from three overlapping partial cDNAs (Yan and Meister, JBC 265: 1588-1593, 1990). The deduced amino acid sequences are 94% similar. Comparison of Northern blots of total RNA isolated from rat kidney or liver with that from human kidney revealed the GCS mRNA to be larger in the human tissue (approximately 4.0 kb vs. approximately 3.7 kb). (The sequence for the human liver GCS cDNA has been assigned accession number M90656 in GenBank/EMBL databases.     

Human Zn-alpha 2-glycoprotein cDNA cloning and expression analysis in benign and malignant breast tissues

Two cDNA clones coding for Zn-alpha 2-glycoprotein (Zn-alpha 2-gp) have been isolated from a human breast library and their nucleotide sequences determined. The deduced amino acid sequence contains the coding information for a hydrophobic signal peptide and the 278 residues of the mature protein. Comparison of this sequence with that from the protein purified from plasma reveals four differences: two amino acid changes (Gln-67 and Glu-222) and insertion of two residues (Ile-75 and Phe-76). Northern-blot analysis showed that the Zn-alpha 2-gp gene is expressed in liver and normal breast, but not in placenta, ovary and thyroid. A comparative analysis in mammary tissues from women with different diseases revealed enhanced expression of Zn-alpha 2-gp gene in benign breast lesions and a variable expression level in breast cancers.     

Rat pineal gland phosducin: cDNA isolation, nucleotide sequence, and chromosomal assignment in the mouse

The pineal gland contains a soluble phosphoprotein, phosducin, which is homologous to that of retinal photoreceptors. Phosducin has been shown to bind the beta, gamma subunits of the retinal G-protein transducin. Retinal phosducin has been cloned and now we report a rat pineal cDNA encoding phosducin. A 1217-nucleotide cDNA was isolated from a rat pineal library by DNA-DNA hybridization with a polymerase chain reaction-amplified cDNA of bovine retina mRNA for phosducin. Northern blot analysis demonstrates that the mRNA for phosducin is approximately 1.3 kb in both rat pineal and rat retina. The translated mRNA from rat pineal encodes a protein with 246 amino acids, compared to the 245 amino acids of bovine retina phosducin. The predicted molecular weight of rat pineal phosducin is 28,201. Immunoblot analysis with affinity-purified antibodies against bovine retina phosducin identify a single immunoreactive protein of approximately 33 kDa in both rat retina and rat pineal. The amino acid sequence of rat pineal phosducin is homologous to that of bovine retina phosducin, revealing 89% identity and another 5.7% similarity. Both rat pineal and bovine retina phosducins are acidic proteins with pIs of 4.3 and 4.5, respectively. The translated protein lacks hydrophobic domains that would suggest an integral membrane protein. Rat pineal phosducin has a single consensus phosphorylation domain for protein kinase A that is nearly identical to that of retinal phosducin, which is phosphorylated by protein kinase A in situ. Rat phosducin also contains three potential phosphorylation domains for protein kinase C and nine for casein kinase II as well as a predicted site for N-glycosylation. The cDNA encoding phosducin was used to localize the gene within a linkage group to a large segment of mouse chromosome 1 in a conserved region with the long arm of human chromosome 1 with a panel of DNA samples from an interspecific cross. In keeping with a proposed role of retinal phosducin in down-regulation of the photo-transduction cascade, a modulatory role in signal transduction is proposed for pineal phosducin.     

Cloning and nucleotide sequence analysis of the human beta-microseminoprotein gene

Beta-microseminoprotein (MSP) is a small protein (94 amino acids) synthesized by the epithelial cells of the prostate gland and secreted into the seminal plasma. Restriction endonuclease mapping of human genomic DNA with a human MSP cDNA probe identified a 19 kilobase (Kb) hybridizing band in both EcoRI and BamHI digestions. Subsequently, the 19 Kb EcoRI fragment of human genomic DNA containing the MSP gene was isolated and cloned into an EMBL4 phage vector. Screening of the recombinant phages resulted in the isolation of one clone containing the MSP gene. Restriction endonuclease mapping and sequence analysis of this clone revealed the human MSP gene of approximately 15 Kb in length. The gene contains four exons and three large introns of approximately 6, 1, and 7 Kb.     

Cloning and nucleotide sequence of a bovine pancreatic preprocarboxypeptidase A cDNA

A full-length cDNA clone encoding bovine pancreatic preprocarboxypeptidase A was isolated and sequenced. The 1405-base pair insert contains a 26-nucleotide 5'-noncoding region, a 1260-nucleotide open reading frame and a 76-nucleotide 3'-noncoding fragment plus a poly(A) tail of at least 43 nucleotides. The open reading frame encodes a protein of 419 amino acids, including the 16 amino acid signal peptide. The mature enzyme (309 residues) has two additional C-terminal amino acids, as compared with the amino acid sequence of the protein which was reported more than 20 years ago. In addition, four residues deduced from the nucleotide sequence differed from those identified in the reported amino acid sequence from their net charge: Asp-89, Asp-114, Gln-122, and Asp-185 instead of Asn-89, Asn-114, Glu-122, and Asn-185, respectively. A high degree of identity exists between the nucleotide sequences (81.3%), on the one hand, and the amino acid sequences (78.3%), on the other hand, of bovine preprocarboxypeptidase A and rat preprocarboxypeptidase A1.     

Cloning and nucleotide sequence of rat ornithine decarboxylase cDNA

The enzyme ornithine decarboxylase (ODC; EC 4.1.1.17) catalyses the first and rate-limiting step in polyamine biosynthesis. Its activity is markedly increased in rapidly growing or regenerating tissue and is subject to regulation by a variety of trophic and mitogenic stimuli. ODC is therefore believed to play an essential role in the onset of cellular proliferation. In a molecular-biological approach to investigate ODC regulation upon induction by tumor promoters in rat liver we isolated an almost full-length rat ODC cDNA clone of 2.4 kb (designated pODC.E10) from a cDNA library of testosterone-induced rat kidney poly(A)+ RNA. Characterization by restriction-endonuclease mapping and sequence analysis showed strong homology to mouse ODC cDNA sequences previously published [Gupta and Coffino, J. Biol. Chem. 260 (1985) 2941-2944; Kahana and Nathans, Proc. Natl. Acad. Sci. USA 82 (1985) 1673-1677; Hickok et al., Proc. Natl. Acad. Sci. USA 83 (1986) 594-598]. This homology is most pronounced in the 461-aa-spanning coding region, amounting to 94% and 97% at the DNA and protein levels, respectively. In the 423-nt 5' leader the rat-mouse homology (approx. 75%) is most pronounced in a region of about 175 nt directly upstream from the translational start site. The leader sequence also contains a perfect inverted repeat of 54 nt and ten additional upstream ATG triplets, which are all followed by nonsense codons before the initiating ATG. In the 633-nt 3' trailer region of pODC.E10 an additional polyadenylation signal is observed more than 300 nt upstream from the 3' end. Rat-mouse homology is about 80% up to this first polyadenylation signal and is considerably less thereafter. The presence of two alternate polyadenylation sites most likely accounts for the 3' size heterogeneity observed in the two ODC mRNAs of 2.1 and 2.6 kb, respectively. In rat liver both mRNAs are coordinately induced by different tumor promoters. Finally, Southern blot analysis of normal rat liver and rat hepatoma DNA revealed that rat ODC, as in other rodents, belongs to a multigene family.     

Cloning and nucleotide sequence of human liver cDNA encoding for cystathionine gamma-lyase.

We have cloned and sequenced a full-length cDNA (1083 bp) encoding the human liver cystathionine-gamma-lyase enzyme (cystathionase). The human cystathionase sequence presented a substantial deletion of 132 bases (44 amino acids) compared to that reported for rat cystathionase, and of 135 bases (45 amino acids) compared to that reported for yeast cystathionase. After re-alignment for the missing nucleotides, the human cDNA sequence shows significant amino acid homology to that for the rat enzyme (85%) and the yeast enzyme (50%). A search for an undeleted cDNA, by the polymerase chain reaction, yielded a second clone which contained the missing 132 bases. Flanking nucleotides in the latter clone were identical to those in the cDNA clone containing the deletion. The two forms of human cystathionase deduced from the two cDNA clones may be derived from two different genes or may be splice variants.