Molecular cloning of human preproacrosin cDNA

Summary Complementary DNA-clones for human preproacrosin have been isolated from a human testis cDNA library in gt11. The nucleotide sequence of the 1402bp cDNA insert includes a 20 bp 5 noncoding region, an open reading frame of 1263bp corresponding to 421 amino acids (45.9 kdalton), and a 105 bp 3 untranslated region. The deduced amino acid sequence is compared with that recently evaluated from a cDNA clone for boar preproacrosin. The sequence identity is 70%; the leader sequence, the catalytic triad (His, Asp, Ser; which is characteristic for serine proteinases) and the positions of the cysteine residues crosslinking the light and the heavy chain of the active enzyme, acrosin, are conserved in both species. At the C-terminal end, a proline-rich sequence is present in both species; this may represent the species-specificity of acrosin.     

Molecular cloning of human apolipoprotein B cDNA.

In this paper we describe the isolation of cDNA clones which code for parts of apolipoprotein B (apoB). The clones were obtained by immunoscreening of an expression library (lambda gt 11) derived from a human hepatoma cell line (Hep G2). The relationship between positive clones and apoB was established with immunochemical techniques using polyclonal as well as monoclonal antibodies. Recombinants, expressing nonoverlapping regions of apoB are described, all hybridizing with a very large mRNA (approximately 20,000 bases long). The nucleotide sequence obtained predicts a primary protein structure with a composition suitable for the formation of stretches of an amphipatic alpha-helix.     

Molecular cloning of cDNA coding for human preprourokinase

A cDNA library was constructed in pBR322 from 18S to 20S mRNA that was extracted from human kidney cells, fractionated on oligo(dT)-cellulose column and sucrose-density gradient, and confirmed for urokinase production in Xenopus laevis oocytes. The Escherichia coli RR1 transformants were hybridized to synthetic oligonucleotide probe prepared according to the known amino acid sequence, Glu 73 to Glu 77 of human urinary urokinase chain B. The entire cloned cDNA covers a 2250-bp region, wherein the 1293-bp sequence codes for preprourokinase consisting of 431 amino acids, with the first 20 residues being a signal peptide. The 5'-untranslated region is at least 80 bp long and the 3'-untranslated region is longer than 850 bp.     

Molecular cloning of cDNA encoding human prointerleukin-6

The data are presented on the cloning and sequencing of cDNA coding for human interleukin-6. The variability of cDNA proIL-6 cloned from different cellular sources was studied. The variability of cDNA proIL-6 may be expressed as heterogeneity of 5'- and 3'-end sequences of cDNA as well as single base-pair changes.     

Molecular cloning of Phaseolus vulgaris cDNA encoding proliferating cell nuclear antigen

A cDNA fragment encoding a common bean (Phaseolus vulgaris) proliferating cell nuclear antigen (PCNA) was isolated using rapid amplification of cDNA 3' end (3' RACE) method, cloned and sequenced. The nucleotide sequence of this clone contains an open reading frame of 798 nucleotides encoding a protein of 265 amino acids. Alignment of the common bean PCNA predicted sequence shows its high degree of identity with PCNA from other plant species. Analysis of PCNA content in the germinating embryos of common bean showed a decrease in the protein level after 60h of germination. Moreover, PCNA was not detected in the tested plant organs (root, stem, leaf and flower). The presence of PCNA in the germinating seeds and its absence from mature plants suggests that this protein plays a crucial role during early stages of plant development.     

Molecular cloning and characterization of cDNA for human myeloperoxidase

Partial amino acid sequence of human myeloperoxidase was determined, and a 41-base oligonucleotide containing deoxyinosines at four positions was chemically synthesized. By using the oligonucleotide as a probe, cDNA clones for human myeloperoxidase were isolated from a cDNA library constructed with mRNA from human promyelocytic leukemia HL-60 cells. One of the clones containing a 2.6-kilobase insert was subjected to nucleotide sequence analysis. The sequence was found to contain an open reading frame, 2,235 nucleotides coding for a protein of 745 amino acids with a calculated Mr of 83,868. The heavy chain of myeloperoxidase, consisting of 467 amino acids, was located on the COOH terminus half of the protein. The RNA specified by the cDNA was prepared using SP6 RNA polymerase and translated in rabbit reticulocyte lysates, and the product was identified as human myeloperoxidase by immunoprecipitation with rabbit anti-human myeloperoxidase antibody. By Northern hybridization analysis of RNA from leukemic cells, it was shown that myeloperoxidase mRNA is abundantly expressed in human promyelocytic HL-60 and mouse myeloid leukemia NFS-60 cells. Furthermore, the results of Southern hybridization analysis of human genomic DNA suggest that there are one or two genes for myeloperoxidase in the human haploid genome.     

Molecular cloning of a cDNA for human delta-aminolevulinate dehydratase

A cDNA encoding human delta-aminolevulinic acid dehydratase (ALA-D; EC 4.2.1.24), the second enzyme in the heme biosynthetic pathway, was isolated from a human liver cDNA expression library. Of the original 17 clones selected with anti-ALA-D antibody, only four expressed anti-ALA-D epitopes as assessed by rescreening with antibody preabsorbed with purified antigen. Subsequent screening of the antibody-positive clones with mixed oligodeoxynucleotide (oligo) probes, synthesized to correspond to human N-terminal and bovine active-site peptide sequences, identified three clones which hybridized only with the oligo probes for the bovine amino acid (aa) sequences. Restriction endonucleases analysis revealed that these three clones contained the same 800-bp cDNA insert. This insert was recloned into bacteriophage M13mp18 and mp19 and sequenced by primer extension. The aa sequence predicted from the partial nucleotide sequence was found to be essentially colinear with the sequences of four bovine ALA-D peptides, totaling 35 non-overlapping aa residues.     

Molecular cloning of cDNA for human prostatic acid phosphatase

A human liver cDNA library in λgt11 was screened with polyclonal antiserum to human acid phosphatase isoenzyme 2a/4. About eleven positive clones have been obtained. Two clones, λ Hap21 and λ Hap22 were further characterized: clone λHap21 contained a 0.8-kb cDNA insert and clone λHap22 a 1.8–2.0-kb insert. XbaI digestion of λHap22 generated two fragments of 1.0 and 0.9 kb. BglII digestion resulted in a 1.2-kb fragment and several smaller fragments of undetermined size. Clone 1 Hap22 contained all the genes carried by λ gt11(lac 5cI857nin 5Sam 100) and the 2-kb insert. An Escherichia coli(λHap22) lysogen was generated, and its acid phosphatase activity was approximately ten-fold higher than that in the control nonlysogenic lysate. Western-blot analysis of total proteins present in this E. coli(λHap22) lysate revealed that the non-induced λHap22 prophage directed the synthesis of an approx. 175-kDa protein. This protein was recognized by antibody to the human acid phosphatase isoenzyme 2a/4 and anti-β-galactosidase and was produced only upon induction with IPTG. These results indicated that AHap22 carried a major portion of the gene coding for the human acid phosphatase isoenzyme 2a and/or 4 and this protein fragment of acid phosphatase was sufficient to manifest enzymatic activity.     

Molecular cloning and sequencing of cDNA encoding for a novel testis-specific antigen

cDNA encoding for a sperm antigen, designated NZ-1, was cloned and sequenced from murine testis cDNA-λgt11 expression library using antibodies to human sperm surface antigens belonging to 14–18 kD molecular region. These sperm antigens are involved in zona pellucida binding and have tyrosine phyosphorylation activity. Computer generated translation analysis of 1395-bp cDNA yielded an open reading frame (ORF) of 152 aa with first ATG, Met start codon at nt 32 and the stop codon TGA at nt 487. The translated protein has a calculated molecular weight of 17.9 kD and a potential tyrosine phosphorylation site at aa 46–54, besides at least two O-linked glycosylation sites. The hydropathy plot generated from the deduced aa sequence indicated it to be a membrane-anchored peptide with a hydrophobic NH₂-terminus that is characteristic of a signal peptide. Extensive computer search in the GenBank, NBRF, and Swiss sequence banks, indicating it to be a novel protein. Northern blot analysis indicated testis-specific expression of NZ-1 antigen. The NZ-1 cDNA was subcloned into pGEX-1λT vector and expressed in glutathione-S-transferase gene fusion system to obtain the recombinant protein. The recombinant protein specifically reacted with the original antibodies raised against the native 14–18 kD sperm proteins. These findings suggest that the sperm-specific recombinant NZ-1 may find applications in the development of a contraceptive vaccine, and in studying the normal and abnormal sperm function and the signal transduction mechanism. Mol. Reprod. Dev. 48:449–457, 1997. © 1997 Wiley-Liss, Inc.     

cDNA coding for the entire human prostate specific antigen shows high homologies to the human tissue kallikrein genes

We have isolated four cDNAs encoding the entire preproprotein of prostate specific antigen from human prostatic cDNA libraries. Comparison of the coding regions of prostate specific antigen with human pancreatic kallikrein (1-3) and human glandular kallikrein (4) showed 73%-84% nucleotide and 61-77% amino acid homologies, respectively, between these enzymes. Also the 3' noncoding regions of these genes were conserved. The close resemblance of prostate specific antigen, a marker for prostatic cancer, to glandular kallikrein suggests related immunogenic properties for them.     

Molecular cloning and sequence analysis of cDNA for human transferrin

A cDNA clone for human transferrin was identified from a human liver cDNA library by pre-screening with different ss-cDNA probes against length-fractionated liver mRNAs, positive hybridization-selection and nucleotide sequence analysis. The insert was of 1 kb, encoding human transferrin from aminoacid 403 through the COOH terminus, with a 3' non coding region of 166 nucleotides. This insert hybridized with a single major mRNA species of about 2.4 kb and several genomic DNA restriction fragments. Hybridization of the Southern blots with different parts of the transferrin insert and at different stringences suggest that the various bands observed correspond to splice sites inside one gene rather than to hybridization to several related genes. Finally, a single or a low number of transferrin gene copies seem to exist in the human genome.     

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.     

Molecular cloning and nucleotide sequence of human pancreatic prechymotrypsinogen cDNA

The cDNA clone encoding human prechymotrypsinogen was isolated from a human pancreas cDNA library and its nucleotide sequence was determined. The sequence consists of a 16 bp 5' non-coding region, a 789 bp amino acid coding region and a 60 bp 3' non-coding region. The predicted product consists of 263 amino acids, including 18 amino acids for a signal peptide and 15 amino acids possible for an activation peptide. Southern blot analyses using the cloned cDNA as a probe revealed that human genomic DNA carries at least two genes that are related to chymotrypsinogen.     

Molecular cloning of the cDNA encoding an immunodominant antigen of Dirofilaria immitis.

An immunodominant antigen of Dirofilaria immitis was studied using recombinant DNA techniques. The mRNA from D. immitis adult female worms was translated in vitro and a major 34 kDa antigenic polypeptide product was demonstrated by immunoprecipitation. cDNA was synthesized from mRNA and a lambda gt11 expression library was constructed and immunoscreened with dirofilariasis positive serum. A positive clone containing a nearly full length cDNA was isolated. The cDNA was 2415 bp in length and consisted of a single open reading frame followed by a long 3' non-coding region of 1446 bp. The open reading frame of 969 bp encoded a polypeptide of 322 amino acids with a molecular weight of 34,400. A cDNA fusion protein synthesized by bacteria (Escherichia coli JM109) using the expression vector pGEMEX-1 was identified as an immunodominant antigen by absorption experiments and had no cross-reactivity with sera from patients with other filarial species.     

Molecular cloning of a cDNA for human pregnancy-specific beta 1-glycoprotein:homology with human carcinoembryonic antigen and related proteins

Human pregnancy-specific beta 1-glycoprotein (SP1) plays an essential role in normal pregnancy. It is also a well-characterized oncodevelopmental antigen, expressed aberrantly by all trophoblastic tumors and some other malignant cell types. Here we report the identification of a human placental cDNA encoding the SP1 polypeptide sequence. The coding sequence shows 95% identity at the nucleotide level with a distinct, recently published SP1 cDNA sequence (PSG16). Unexpectedly, the sequence is also highly homologous to the published sequence of human carcinoembryonic antigen (CEA). SP1, CEA and CEA-related nonspecific cross-reacting species thus belong to a group of closely related though antigenically diverse tumor-associated glycoproteins. Comparison of the deduced amino acid sequence of the SP1 cDNA with that of CEA provides insight into the modular nature of these related proteins. This may have implications for the genomic organization and evolution of the CEA gene family.