Increased messenger RNA for allograft inflammatory factor-1, LERK-5, and a novel gene in 17.5-day relative to 15.5-day bovine embryos

Considerable embryonic loss occurs between Gestation Days 15 and 18 in cattle when critical cellular and molecular events occur, including maternal recognition of pregnancy. To gain insight into these events, mRNA differential display analysis was used to identify eight unique cDNA fragments present in greater abundance in 17.5-day than in 15.5-day bovine embryos. Four cDNA fragments, confirmed to be upregulated in 17.5-day embryos using Northern analysis, were cloned and sequenced. Three cDNA fragments shared sequence identities with known homologs: human allograft inflammatory factor-1 (AIF-1), human LERK-5, and bovine interferon-tau. One novel cDNA fragment did not share sequence identity to previously reported genes, except for a similar DNA sequence in the human genome. AIF-1 mRNA was present in developing placenta through Gestation Day 36, and abundant levels were observed in adult bovine spleen and lung. The novel gene, which we have named periattachment factor (PAF), was not detected in adult tissues using Northern analysis or in conceptuses between Days 30 and 36 of pregnancy. Additional sequence information for bPAF was obtained from a cDNA library constructed from a 25-day bovine embryo. The protein corresponding to the open reading frame has four protein kinase C phosphorylation sites, two casein kinase II phosphorylation sites, a nuclear targeting sequence, but no obvious DNA or RNA binding motifs. Abundant expression of this gene during a narrow but critical window of embryonic development makes it worthy of further study.     

A form of human basic fibroblast growth factor with an extended amino terminus

The amino acid sequence of a human placental bFGF was determined by a combination of protein and cDNA sequencing. The placental bFGF consists of 157 amino acid residues with a calculated molecular weight of 17,464 and is highly homologous to bovine pituitary bFGF. The human protein contains an amino terminal extension when compared to the sequence established for bovine bFGF (Esch et al., 1985) and to the sequence of the predicted translation product based on human bFGF cDNA clones (Abraham et al., 1986).     

Primary structure of cDNA for bovine beta-casein

In this work the complete sequence of 1097 nucleotide bovine beta-casein cDNA has been determined. Sequencing of end labeled fragments was performed using the method of Maxam and Gilbert. Bovine beta-casein cDNA consist of a 672 nucleotide coding region, flanked by 60 nucleotide 5' and 358 nucleotide 3'-noncoding region. The restriction map of beta-casein cDNA was constructed. The computer analysis was done and the comparisons of the complete sequence of bovine beta-casein cDNA with other sequences, coding caseins in bovine, rat and guinea-pig was performed. It was determined, that cloned cDNA is related to genetic variant A1.     

A novel cDNA clone encoding a prolactin-like protein that lacks the two C-terminal cysteine residues isolated from bovine placenta

A new prolactin-like cDNA clone, bPLP-IV, was isolated from a bovine placental cDNA library and the complete nucleotide sequence was determined. The bPLP-IV encodes a protein consisting of 237 amino acids, which is related to, but different from seven other known bovine prolactin-like proteins including two placental lactogens. The predicted amino acid sequence of the bPLP-IV shows over 52% identity to other known members of bovine prolactin-like proteins, 48% to bovine prolactin, 40% to both two bovine placental lactogens and only 22% to bovine growth hormone. The bPLP-IV protein has a unique feature in its primary structure, lacking the two C-terminal cysteine residues which are completely conserved in all other known members of prolactin-growth hormone-placental lactogen gene family. The expression of bPLP-IV in developing bovine placenta was apparently stage-specific, being maximal in the full-term placenta.     

Molecular cloning and sequence analysis of cDNA encoding human prostatic acid phosphatase

lambda gt11 clones encoding human prostatic acid phosphatase (PAP) (EC 3.1.3.2) were isolated from human prostatic cDNA libraries by immunoscreening with polyclonal antisera. Sequence data obtained from several overlapping clones indicated that the composite cDNAs contained the complete coding region for PAP, which encodes a 354-residue protein with a calculated molecular mass of 41,126 Da. In the 5'-end, the cDNA codes for a signal peptide of 32 amino acids. Direct protein sequencing of the amino-terminus of the mature protein and its proteolytic fragments confirmed the identity of the predicted protein sequence. PAP has no apparent sequence homology to other known proteins. However, both the cDNA clones coding for human placental alkaline phosphatase and PAP have an alu-type repetitive sequence about 900 nucleotides downstream from the coding region in the 3'-untranslated region. Two of our cDNA clones differed from others at the 5'-ends. RNA blot analysis indicated mRNA of 3.3 kb. We are continuing to study whether acid phosphatases form a gene family as do alkaline phosphatases.     

Molecular cloning and characterization of cDNA encoding mouse cytokeratin no. 19

We have isolated cDNA clones encoding the mouse cytokeratin No. 19 (Ck 19) from an intestinal cDNA library using synthetic oligodeoxyribonucleotides as probes. We obtained four independent clones, which correspond to about 1.4-kb of ck19 cDNA. Nucleotide sequence analysis revealed that these cDNAs encode a protein of 44,541 Da composed of 403 amino acids (aa). The deduced aa sequence defines an alpha-helical central domain, and suggests that the protein lacks a C-terminal non-alpha-helical tail segment, characteristic of the human and bovine 40-kDa keratins (Ck19). The overall aa identity between mouse Ck19 and human and bovine Ck19 is very high, 82.7% and 82.4%, respectively. The coil-forming central domain of mouse Ck19 has 45-65% similarity to other type-I Ck polypeptides, while it displays only 20-30% similarity to type-II Ck polypeptides. Northern blot analysis showed that mouse ck19 mRNA is strongly expressed in adult intestine, stomach and uterus. Interestingly, it is expressed in a placental cell line and a retinoic acid-treated mouse teratocarcinoma cell line (F9), but not in a parietal yolk sac endoderm-like cell line (PYS-2). This pattern of expression is very similar to that for the mouse gene encoding extra-embryonic endodermal cytoskeletal protein C (EndoC), suggesting they may be the same.     

Molecular cloning and sequence analysis of human placental alkaline phosphatase

The complete amino acid sequence of the precursor and mature forms of human placental alkaline phosphatase have been inferred from analysis of a cDNA. A near full-length PLAP cDNA (2.8 kilobases) was identified upon screening a bacteriophage lambda gt11 placental cDNA library with antibodies against CNBr fragments of the enzyme. The precursor protein (535 amino acids) displays, after the start codon for translation, a hydrophobic signal peptide of 21 amino acids before the amino-terminal sequence of mature placental alkaline phosphatase. The mature protein is 513 amino acids long. The active site serine has been identified at position 92, as well as two putative glycosylation sites at Asn122 and Asn249 and a highly hydrophobic membrane anchoring domain at the carboxyl terminus of the protein. Significant homology exists between placental alkaline phosphatase and Escherichia coli alkaline phosphatase. Placental alkaline phosphatase is the first eukaryotic alkaline phosphatase to be cloned and sequenced.     

Cloning of the authentic bovine gene encoding pepsinogen a and its expression in microbial cells

Bovine pepsin is the second major proteolytic activity of rennet obtained from young calves and is the main protease when it is extracted from adult animals, and it is well recognized that the proteolytic specificity of this enzyme improves the sensory properties of cheese during maturation. Pepsin is synthesized as an inactive precursor, pepsinogen, which is autocatalytically activated at the pH of calf abomasum. A cDNA coding for bovine pepsin was assembled by fusing the cDNA fragments from two different bovine expressed sequence tag libraries to synthetic DNA sequences based on the previously described N-terminal sequence of pepsinogen. The sequence of this cDNA clearly differs from the previously described partial bovine pepsinogen sequences, which actually are rabbit pepsinogen sequences. By cloning this cDNA in different vectors we produced functional bovine pepsinogen in Escherichia coli and Saccharomyces cerevisiae. The recombinant pepsinogen is activated by low pH, and the resulting mature pepsin has milk-clotting activity. Moreover, the mature enzyme generates digestion profiles with alpha-, beta-, or kappa-casein indistinguishable from those obtained with a natural pepsin preparation. The potential applications of this recombinant enzyme include cheese making and bioactive peptide production. One remarkable advantage of the recombinant enzyme for food applications is that there is no risk of transmission of bovine spongiform encephalopathy.     

Identification of a cDNA clone for the beta-subunit of the pyruvate dehydrogenase component of human pyruvate dehydrogenase complex

We report the isolation of a 1.5 kb cDNA clone for the beta subunit of human pyruvate dehydrogenase (E1) from a human liver lambda gt11 cDNA library using anti-E1 serum. We generated a peptide sequence of 24 amino acids starting from the N-terminus of bovine heart mature E1 beta. The identity of the E1 beta cDNA clone was confirmed by the similarity between the amino acid sequence deduced from the cDNA nucleotide sequence and the known amino acid sequence of bovine heart E1 beta. In Northern analysis of total RNA extracted from human heart, the E1 beta cDNA clone hybridized to a major 1.6 kb and a minor 5.2 kb RNA species.     

Complete primary structure of the transacylase (E2b) subunit of the human branched chain alpha-keto acid dehydrogenase complex

We isolated from a placental cDNA library by immunoscreening a cDNA clone encoding the transacylase (E2b) precursor of the human branched chain alpha-keto acid dehydrogenase (BCKDH) complex. The cDNA insert consists of 2,649 base pairs with an open reading frame of 1,431 base pairs which can be translated into 477 amino acids and a 3'-untranslated region of 1,205 base pairs. The deduced amino acid sequence includes a leader peptide of 56 amino acid residues, a lipoyl-bearing domain, a E3-binding domain and an inner core domain. A mature human E2b subunit is likely to contain 421 amino acid residues with a calculated Mr 46,322. The nucleotide sequence of the open reading frame and the deduced amino acid sequence of the human E2b shows 91.6% and 92.0% homology with those of the bovine E2b subunit, respectively.     

Molecular cloning and sequence analysis of human placental ferredoxin

We have characterized several clones specific for the human iron-sulfur protein, ferredoxin, which is involved in electron transfer to mitochondrial cytochromes P-450. Clones were isolated from a human placental cDNA expression library in lambda gt11 by immunoscreening with antibody to bovine adrenal ferredoxin. One clone contained the entire amino acid coding sequence (552 bp) together with 27 bp at the 5'-terminus and approximately 0.9 kb at the 3'-terminus; this form appears to correspond to the major mRNA species of approximately 1.7 kb observed on Northern blots of placental mRNA. The deduced amino acid sequence suggests that human ferredoxin is synthesized as a precursor of 184 amino acids (Mr 19,371) which is cleaved to yield a polypeptide of 124 amino acids (Mr 13,546). The mature protein is highly acidic, and the sequence is very similar to those of bovine and porcine adrenodoxins with the exception of substitutions and variations in length at the C-terminus. The N-terminal precursor segment, on the other hand, is considerably diverged from that determined for bovine adrenodoxin, but is similar in overall basicity and the pattern of occurrence of arginine residues.     

Hybridization and partial cDNA sequence analyses of bovine lung angiotensin I-converting enzyme

The mRNA encoding angiotensin I-converting enzyme, a zinc-metallo dipeptidyl carboxyhydrolase, has been identified in extracts prepared from bovine lung tissue. Bovine lung poly(A) + mRNAs were subjected to electrophoresis and northern blot hybridization analysis using a radiolabeled synthetic 24-deoxyoligonucleotide probe complementary to eight codons for amino acids at the active-site of the enzyme (Harris, R.B. & Wilson, I.B., J. Biol. Chem. 260, 2208-2211, 1985). This amino acid sequence contains the catalytic glutamic acid residue. A single RNA species (approximately equal to 4 kb) was detected which is 1 kb larger than predicted from the molecular weight of the enzyme. The excess nucleic acid composition may be due to leader and/or trailer sequences or the RNA may encode a high molecular weight precursor form of the enzyme. We have cloned an EcoR1-HindIII digest fragment (1400 bp) of the duplex cDNA derived from the bovine lung converting enzyme poly(A) + mRNA and also Bal31 deletion fragments generated from the 1400 bp clone. Several of the Bal31 clones contain the active-site sequence codons of the enzyme and the complete cDNA sequence of one of these (72 bp) has been determined. We found the amino acid sequence at the active site to be -Phe-Thr-Glu-Leu-Ala-Asn-Ser-, containing the catalytic Glu residue. This sequence is identical with the sequence that we previously determined by manual Edman degradation analysis of the appropriate active-site peptide except that we now find Asn instead of Asp. We have sequenced 670 bp of the 1400 bp clone but have not yet overlapped the active-site sequence.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of gene expression profiles in early bovine pregnancy using a custom cDNA microarray

Gene expression analysis comparing nonpregnant with pregnant bovine uteri, including placenta, was performed with a custom cDNA microarray containing 1,933 independent genes. These genes were classified into six categories according to biological function, as follows: cell and tissue structural dynamics (108 genes), intercellular communication (221), intracellular metabolism (265), cell cycle and apoptosis (26), regulation of gene expression (113), expressed sequence tag (EST) and function unknown (617), and uncomplemented genes (583 clones). This array possessed bovine placental/endometrial specificity, as it included many pregnancy-specific molecules, such as pregnancy-associated glycoprotein-1 (PAGs), placental lactogen (PLs), and prolactin-related protein-1 (PRPs). A total of 77 genes were induced and 12 repressed in the placenta/endometrium. Our results point to a fundamental role for bovine placental-specific genes such as PAGs, PLs, and PRPs, in implantation and placentogenesis, and document that cDNA microarray analysis from bovine placenta/endometrium is possible and is a specific tool for monitoring genome-wide gene expression during the establishment and maintenance of pregnancy.     

Characterization and Expression of the cDNA Coding for the Human Myelin/Oligodendrocyte Glycoprotein

Abstract: We report here the characterization of a full-length cDNA encoding the human myelin/oligodendrocyte glycoprotein (MOG). The sequence of the coding region of the human MOG cDNA is highly homologous to that of other previously cloned mouse, rat, and bovine MOG cDNAs, but the 3' untranslated region differs by an insertion of an Alu sequence between nucleotides 1,590 and 1,924. Accordingly, northern blot analyzes with cDNA probes corresponding to the coding region or the 3' untranslated Alu-containing sequence revealed a single band of 2 kb, rather than the 1.6 kb of bovine, rat, or mouse MOG cDNA(s). Immunocytochemical analysis of HeLa cells transfected with human MOG cDNA, which was performed using a specific antibody raised against whole MOG, clearly indicated that MOG is expressed at the cell surface as an intrinsic protein. These data are in accordance with the predicted amino acid sequence, which contains a signal peptide and two putative transmembrane domains. The knowledge of the human MOG sequence should facilitate further investigations on its potential as a target antigen in autoimmune demyelinating diseases like multiple sclerosis.     

Cloning and characterization of a cDNA coding for the lipoprotein-associated coagulation inhibitor shows that it consists of three tandem Kunitz-type inhibitory domains

Human plasma contains a lipoprotein-associated coagulation inhibitor (LACI) which inactivates factor Xa directly, and in a Xa-dependent fashion also inhibits the VIIa-tissue factor complex of the extrinsic coagulation pathway. Rabbit polyclonal anti-LACI antiserum was used to screen human placental and fetal liver lambda gt11 cDNA libraries for the expression of LACI antigens. Immunologically positive clones were further tested for their ability to bind 125I-factor Xa. Seven clones were obtained which are immunologically and functionally active. The longest cDNA insert (lambda P9) of these isolates is 1.4 kilobases (kb) while other clones are 1.0 kb in length. Nucleotide sequence analysis shows that lambda P9 consists of 1431 bases that include a 5'-noncoding sequence of 132 nucleotides, an open reading frame of 912 nucleotides, and a 3'-noncoding region of 387 nucleotides. The open reading frame encodes a signal peptide of 28 residues followed by a 32-kilodalton protein of 276 residues. The predicted sequence of mature LACI contains 18 cysteines and three potential N-linked glycosylation sites. The amino acid sequence analysis of purified LACI's NH2 terminus and two of its proteolytic fragments match exactly those deduced from the cDNA sequence, indicating that the cDNA codes for LACI. The translated amino acid sequence of LACI shows several discernible domains, including a highly negatively charged NH2 terminus, three tandem Kunitz-type inhibitory domains, and a highly positively charged carboxyl terminus. Northern blot analysis shows that the following liver-derived cell lines, Chang liver, HepG2 hepatoma, and SK hepatoma all, contain two major species of mRNA (1.4 and 4.4 kb) which hybridize with LACI cDNA.     

Cloning of the Authentic Bovine Gene Encoding Pepsinogen A and Its Expression in Microbial Cells

Bovine pepsin is the second major proteolytic activity of rennet obtained from young calves and is the main protease when it is extracted from adult animals, and it is well recognized that the proteolytic specificity of this enzyme improves the sensory properties of cheese during maturation. Pepsin is synthesized as an inactive precursor, pepsinogen, which is autocatalytically activated at the pH of calf abomasum. A cDNA coding for bovine pepsin was assembled by fusing the cDNA fragments from two different bovine expressed sequence tag libraries to synthetic DNA sequences based on the previously described N-terminal sequence of pepsinogen. The sequence of this cDNA clearly differs from the previously described partial bovine pepsinogen sequences, which actually are rabbit pepsinogen sequences. By cloning this cDNA in different vectors we produced functional bovine pepsinogen in Escherichia coli and Saccharomyces cerevisiae. The recombinant pepsinogen is activated by low pH, and the resulting mature pepsin has milk-clotting activity. Moreover, the mature enzyme generates digestion profiles with α-, β-, or κ-casein indistinguishable from those obtained with a natural pepsin preparation. The potential applications of this recombinant enzyme include cheese making and bioactive peptide production. One remarkable advantage of the recombinant enzyme for food applications is that there is no risk of transmission of bovine spongiform encephalopathy.     

Identification of bacterial clones encoding bovine caseins by direct immunological screening of the cDNA library

A sensitive immunoassay was used to identify recombinant plasmids carrying cDNA fragments of bovine caseins in the cDNA library from bovine mammary gland mRNA. Colonies grown on nitrocellulose filters were lysed in situ and proteins from the lysates were blotted onto CNBr-activated cellulose filter paper. Antigens covalently bound to CNBr-activated paper or bound to nitrocellulose filters were detected by reaction with antiserum to caseins, followed by ¹²⁵I-labelled Staphylococcus aureus protein A and autoradiography. Six clones were found positive among 5400 of the cDNA library: 3-A1, 3-B2, 3-B5, 3-H7, 2-A5 and 2-C9. The molecular weights of chimeric pre-β-lactamase: casein proteins synthesized in Escherichia coli were estimated by immunoblotting. Colony hybridization and nucleotide sequence analysis showed that clone 3-B5 contained a cDNA fragment of bovine χ-casein, clone 3-H7 contained a cDNA fragment of β-casein, while clones 2-A5 and 2-C9 carried cDNA fragments of α_si-casein.