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N E Cooke

Publications and source records attributed to N E Cooke.

At least 55 records · Page 3Linked to original sources
PubMed1989

The vitamin D-binding protein, alpha-fetoprotein, albumin multigene family: detection of transcripts in multiple tissues.

The serum vitamin D-binding protein (DBP), a member of a multigene family that includes alpha-fetoprotein (alpha FP) and albumin (ALB), is expressed at high levels in the adult liver. Although several studies have demonstrated the presence of DBP on the cell surface of immunocytes and cytotrophoblasts, evidence of extra-hepatic DBP synthesis remains inconclusive. Using Northern blot analysis of RNA from multiple rat tissues, the DBP transcripts were present in the expected high levels in the adult liver. In addition, DBP mRNA was found in the adult kidney, testis, abdominal fat, and 18-day fetal yolk sac. The concentrations of DBP mRNA in these tissues is 100-1000-fold less than in the liver. Polymerase chain reaction amplification (PCR) of DBP, alpha FP, and ALB cDNAs, reverse transcribed from a wide variety of rat tissue RNA samples, was performed to examine their respective tissue-specific patterns of gene transcripts. The tissues in which DBP gene transcripts were noted by Northern analysis were paralleled at 20 cycles of PCR with liver much greater than kidney, yolk sac, testis, and abdominal fat. Similarly at 20 cycles, alpha FP-amplified fragments were detected in the yolk sac much greater than brain, testes, uterus, liver, and placenta; and ALB-amplified fragments in liver much greater than testis, uterus, placenta, and yolk sac. Unexpectedly, at 32 cycles of PCR, DBP, alpha FP, and ALB mRNAs could be detected at very low levels in all tissues examined. To examine the nature of the DBP mRNA from these nonhepatic sources, a Sprague-Dawley kidney cDNA library was screened, and a single cDNA recombinant was identified. This clone contained a full length DBP cDNA with 14 nucleotide and 3 predicted amino acid differences from the Fischer liver DBP cDNA reported previously. We conclude that the kidney and liver DBP mRNAs are transcription products of the same DBP locus and that DBP, alpha FP, and ALB genes display different spectra of tissue distribution. The unexpected finding that all three spliced RNA transcripts could be detected in at least trace quantities in all tissues studied suggests a leaky level of transcription by this multigene family or their transcription at low levels by an unidentified stromal cell common to all organs studied.

Amino Acid Sequence↗
PubMed1989

Characterization and histologic localization of human growth hormone-variant gene expression in the placenta.

The human growth hormone-variant (hGH-V) gene is one of five highly similar growth hormone-related genes clustered on the short arm of chromosome 17. Although the pattern of expression of the adjacent normal growth hormone (hGH-N) and chorionic somatomammotropin (hCS) genes in this cluster are well characterized, the expression of the hGH-V gene remains to be defined. In previous studies, we have demonstrated that the hGH-V gene is transcribed in the term placenta and expressed as two alternatively spliced mRNAs: one is predicted to encode a 22-kD hormone (hGH-V), the other retains intron 4 in its sequence resulting in the predicted synthesis of a novel 26-kD hGH-V-related protein (hGH-V2). In the present report, we document the expression of both of these hGH-V mRNA species in the villi of the term placenta, demonstrate an increase in their concentrations during gestation, and directly sublocalize hGH-V gene expression to the syncytiotrophoblastic epithelium of the term placenta by in situ cDNA-mRNA histohybridization. The demonstrated similarity in the developmental and tissue-specific expression of the hGH-V gene with that of the related hCS gene suggests that these two genes may share common regulatory elements.

Animals↗
PubMed1989

Glycosylated human growth hormone variant.

The human growth hormone variant (hGH-V) gene is expressed by the syncytiotrophoblastic layer of the human placenta in two forms: hGH-V mRNA encoding a 22 kD protein, and hGH-V2 mRNA which retains intron 4 and is expected to encode a 26 kD protein. There is a predicted N-linked glycosylation site in hGH-V at amino acid 140 that is absent in both hGH-V2 and in the highly homologous normal pituitary GH (hGH-N). Cell lines transfected with the hGH-N gene secrete 22 kD GH and the 20 kD product of an alternatively spliced mRNA, while cell lines transfected with the hGH-V gene secrete three proteins of 22, 24, and 26 kD. To determine whether any of these hGH-V isoforms are glycosylated, the cell lines were grown in the absence and presence of tunicamycin. In addition, conditioned medium from metabolically labelled hGH-V transfected cells was separately digested with peptide:N-glycosidase F and endoglycosidase H. The 26 and 24 kD bands were both absent from the media after tunicamycin treatment and were both sensitive to peptide:N-glycosidase F treatment. Endoglycosidase H digestion resulted in the selective loss of the 24 kD band. These results indicate that hGH-V is partially modified posttranslationally by N-linked glycosylation in a fibroblastic cell line.

Animals↗
PubMed1988

Two distinct species of human growth hormone-variant mRNA in the human placenta predict the expression of novel growth hormone proteins.

We have sought direct evidence for the in vivo expression of the human growth hormone-variant (hGH-V) gene by screening a placental cDNA library with a hGH-V-specific oligonucleotide. Nine independent hGH-V cDNA clones were isolated and analyzed, and three distinct species were detected. Five of these hGH-V cDNAs represent mRNAs spliced and processed in a pattern analogous to that of the highly homologous human growth hormone and human chorionic somatomammotropin gene transcripts. Each of the remaining four hGH-V cDNAs contains an additional segment of 253 nucleotides corresponding in position and sequence to the fourth intron of the hGH-V gene. In addition, one of the mRNAs in this second group uses an alternative downstream polyadenylation site. The alternatively spliced hGH-V mRNA, which we refer to as hGH-V2 mRNA, constitutes approximately 30% of the hGH-V transcripts both in the human term placenta and in a stable mouse fibroblast line expressing the transfected hGH-V gene. The placental expression of the hGH-V gene is specific to villous tissue. The hGH-V2 mRNA is predicted to encode a protein which substitutes the 65 carboxyl-terminal amino acids of hGH-V with a new 104-residue carboxyl terminus resulting in significant divergence in their relative physical properties. The alternative splicing of the hGH-V transcripts to hGH-V and hGH-V2 mRNAs expands the potential complexity of the hGH-V gene's role in normal placental function.

Amino Acid Sequence↗
PubMed1988

Human growth hormone gene and the highly homologous growth hormone variant gene display different splicing patterns.

Stably transfected cell lines containing the normal human growth hormone (hGH-N) and human growth hormone-variant (hGH-V) genes have been established in order to study the expression of these two highly homologous genes. Each gene was inserted into a bovine papillomavirus shuttle vector under the transcriptional control of the mouse metallothionein gene promoter and the resultant recombinants were transfected into mouse C127 cells. The transfected cells containing the hGH-N gene secrete two hGH proteins, 91% migrating at 22 kD and 9% migrating at 20 kD, the same relative proportions synthesized in vivo by the human pituitary. S1 nuclease analysis of mRNA from these cells confirms that 20 kD hGH is encoded by an alternatively spliced product of the primary hGH-N gene transcript in which the normal exon 3 splice-acceptor site is bypassed for a secondary site 15 codons within exon 3. Although the hGH-V gene is identical to the hGH-N gene for at least 15 nucleotides on either side of the normal and alternative exon 3 AG splice-acceptor sites, hGH-V synthesizes only a 22-kD protein. Reciprocal exchange of exon 3 and its flanking intron sequences between the hGH-N gene and the hGH-V gene, eliminates the synthesis of the 20-kD protein in both resultant chimeric genes. These results directly demonstrate that both the major 22-kD and the minor 20-kD forms of pituitary hGH are encoded by the alternative splicing products of a single hGH-N gene transcript. This alternative splicing is neither species nor tissue-specific and appears to be regulated by at least two separate regions remote from the AG splice-acceptor site.

Animals↗
PubMed1986

Synthesis of growth hormone-prolactin chimeric proteins and processing mutants by the exchange and deletion of genomic exons.

To test the feasibility of synthesizing recombinant peptide hormones by exon deletion and exchange, we have constructed and expressed hybrid human growth hormone (hGH)-rat prolactin (rPrl) genes in which the third and fourth exons of the hGH gene are deleted and separately replaced by the corresponding exons of the rPrl gene. These exon deletion and exon exchange genes were inserted into an SV40 viral vector, packaged, and expressed following acute viral infection of monkey kidney cells. Expression of the hGH gene lacking the third exon (hGHd3) was not detectable at the level of protein production. However, replacement of the deleted third exon in the hGHd3 gene with exon 3 of the rPrl gene (hGHP3) resulted in the efficient synthesis of a secreted chimeric protein. When the fourth exon of the hGH gene was deleted (hGHd4), the encoded protein was found only in the cytosol despite signal sequence processing. Replacement of the deleted fourth exon in this hGHd4 gene with exon 4 of rPrl resulted in the synthesis and secretion of both a chimeric protein (hGHP4) as well as a larger protein corresponding in size to prehGHP4. These results suggest that exon exchange among distantly related genes in the GH family may be used to produce high levels of chimeric GH-related proteins, and regions internal to the hGH protein may be critical in establishing normal protein processing and secretion.

Chimera↗
PubMed1986

Rat vitamin D binding protein. Determination of the full-length primary structure from cloned cDNA.

Vitamin D binding protein (DBP) is an abundant serum glycoprotein secreted by the liver which transports vitamin D sterols, binds to actin, and is found on the surface of B-lymphocytes and subpopulations of T-lymphocytes. In the current study, a cDNA to rat DBP mRNA was cloned from a bacteriophage lambda gt 11 rat liver expression library. This DBP cDNA clone was identified by immunoblotting and its identity was confirmed by immunoprecipitation of a 54-kDa protein after hybrid-assisted translation. Northern analysis and primer extension mapping of rat liver mRNA indicated that the full-length DBP mRNA contains 1700 bases. By DNA sequence analysis this 1655-base pair clone contains a single open reading frame encoding the 476-amino acid containing full-length DBP and includes its 16-amino acid signal sequence. Analysis of the sequence reveals about 40% nucleotide and 23% amino acid homology to both albumin and alpha-fetoprotein. The encoded DBP contains a characteristic placement of cysteine residues, identical to that in albumin, suggesting a similar secondary folding structure. Albumin and alpha-fetoprotein are composed of three internally homologous domains. DBP mRNA terminates 122 amino acids before the larger albumin mRNA in the third internal domain, but retains the characteristic homology among the first two domains and the truncated portion of the third domain. These data support the conclusion that DBP is a member of a multigene family which includes albumin and alpha-fetoprotein.

Albumins↗
PubMed1986

Direct regional assignment of the gene for vitamin D binding protein (Gc-globulin) to human chromosome 4q11-q13 and identification of an associated DNA polymorphism.

Using a characterized human vitamin D binding protein (DBP) cDNA probe and a panel of rodent X human somatic cell hybrids, we established the chromosomal location of the structural gene for DBP on human chromosome 4. In situ hybridization of 3H-labeled DBP cDNA to human metaphase chromosomes confirmed this assignment and allowed regional localization to bands 4q11-4q13. A restriction fragment length polymorphism associated with the DBP gene that should prove useful in future linkage studies was identified with the enzyme BamHI.

Alleles↗
PubMed1986

The related genes encoding growth hormone and prolactin have been dispersed to chromosomes 10 and 17 in the rat.

We have assigned the rat GH gene to chromosome 10 and the rat PRL gene to chromosome 17. DNA from a series of mouse BWTG3 x rat hepatocyte somatic cell hybrids, each of which has retained a unique complement of rat chromosomes, was analyzed for the presence of rat GH and PRL genomic fragments by Southern blotting. Radiolabeled complementary DNAs (cDNAs) encoding rat GH and rat PRL were used as molecular probes. Based upon these assignments, we conclude that the evolutionarily related GH and PRL genes have been dispersed to different chromosomes in rat as in man.

Animals↗
PubMed1985

Serum vitamin D-binding protein is a third member of the albumin and alpha fetoprotein gene family.

A near full-length cDNA encoding the human vitamin D-binding protein (hDBP) was isolated from a human liver mRNA expression library. Complete sequence analysis of this clone predicts the full-length amino acid sequence of the pre-hDBP. Comparison of the sequence of the hDBP mRNA and protein to existing protein and nucleic acid data banks demonstrates a strong and highly characteristic homology of the hDBP with human albumin (hALB) and human alpha-fetoprotein (hAFP). Based upon this structural comparison, we establish that DBP is a member of the ALB and AFP gene family.

Amino Acid Sequence↗
PubMed1981

The prolactin gene is located on chromosome 6 in humans.

The gene for prolactin has been located on chromosome 6 in humans. DNA fragments of 4.8 and 4.0 kilobases containing prolactin gene sequences were identified in human genomic DNA, whereas DNA fragments of 7.4, 3.6, and 3.3 kilobases containing prolactin gene sequences were found in mouse cells. In somatic cell hybrids of human and mouse cells the 7.4-, 3.6-, and 3.3-kilobase mouse fragments were always present, whereas the 4.8- and 4.0-kilobase human fragments were only present when human chromosome 6 was also present. We conclude that the prolactin gene resides on chromosome 6, a different location from those of the genes for the related hormones chorionic somatomammotropin and growth hormone.

Animals↗
PubMed1981

Human prolactin. cDNA structural analysis and evolutionary comparisons.

Prolactin (Prl), growth hormone, and chorionic sommatomammotropin form a set (the "Prl set") of hormones which is thought to have evolved from a common ancestral gene. This assumption is based on several lines of evidence: overlap in their biological and immunological properties, similarities in their amino acid sequences, and homologies in the nucleic acid sequences of their structural genes. In the current study we report the cloning, amplification in bacteria, and sequence analysis of DNA complementary to Prl mRNA isolated from human pituitary Prl-secreting adenomas. The cloned DNA contains 914 bases, which includes the entire coding sequence of human prePrl as well as portions of the 5- and 3'-untranslated regions of the mRNA. The amino acid sequence predicted by our data differs from a previously reported amino acid sequence in 8 positions. With the results of this study we can now compare in one species the nucleotide sequences of the structural gene coding for each of the hormones of the Prl set. The sequence divergence at replacement sites is used to establish an evolutionary clock for the Prl set of genes. Using this clock, we postulate that the chromosomal segregation of human Prl and human growth hormone occurred about 392 million years ago and that growth hormone and chorionic sommatomammotropin underwent an intrachromosomal recombination within the last 10 million years.

Adenoma↗