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N E Cooke

Publications and source records attributed to N E Cooke.

At least 19 recordsLinked to original sources
PubMed1996

Electrophoretic mobility shift assay identifies vitamin D binding protein (Gc-globulin) in human, rat, and mouse sera.

Serum vitamin D binding protein (DBP, also known as Gc-globulin) is a multifunctional protein capable of binding both vitamin D metabolites and actin. DBP can be visualized when analyzed by polyacrylamide gel electrophoresis followed by staining. Confirmation of its identity had previously required immunoprecipitation with specific anti-DBP antisera or occupancy of the protein with radioactive vitamin D sterols. We present studies showing that preincubation of G-actin with mammalian sera produced a discernible DBP protein band shift on native gel electrophoresis. Addition of DNaseI, a 33-kDa intracellular protein with an avid actin-binding site, to the incubations resulted in a supershift of DBP-actin complexes to an even more cathodal region of the gels. Following incubations with human, rat, and murine sera the same actin shift occurred as did the actin plus DNaseI supershift. The migrations of each complex were correlated with purified DBP migrations under identical conditions. It was confirmed that the supershifted bands contained DBP by Western blotting and detection of DBP by binding of 25-OH[3H]D3. After intravenous G-actin injections into living mice, a serum DBP-actin complex could be detected on native gels as the uncomplexed DBP band decreased in intensity. This simple, direct-staining technique appears to be suitable for identifying DBP/Gc phenotypes in human populations as well as for semiquantitatively monitoring the plasma actin-scavenger system in vivo in animal models or in human diseases.

Actins↗
PubMed1995

Physical linkage of the human growth hormone gene cluster and the skeletal muscle sodium channel alpha-subunit gene (SCN4A) on chromosome 17.

The human growth hormone (GH) locus, a cluster of five genes, spans 47 kb on chromosome 17q22-q24. The skeletal muscle sodium channel alpha-subunit locus (SCN4A), a 32.5-kb gene, has previously been mapped to 17q23.1-q25.3. We demonstrate that both the GH gene cluster and the SCN4A gene colocalize to a single 525-kb yeast artificial chromosome (YAC) containing DNA derived from human chromosome 17. Restriction maps of two cosmids encompassing the 5' terminus of the GH locus and including up to 40 kb of 5'-flanking sequences demonstrate a perfect 20-kb overlap with previously published maps of the SCN4A gene. A 720-bp DNA segment, encompassing sequences 32.3 to 31.6 kb 5' to GH, was sequenced and found to be identical to exon 14 of SCN4A. These data demonstrate that the SCN4A gene and the entire GH gene cluster are contained within 100 kb on chromosome 17 and are separated by only 21.5 kb. Remarkably, this physical linkage between GH and SCN4A also reveals that multiple elements critical to tissue-specific transcriptional activation of the GH gene lie within the SCN4A gene.

Base Sequence↗
PubMed1995

The human growth hormone gene is regulated by a multicomponent locus control region.

The five-member human growth hormone (hGH)/chorionic somatomammotropin (hCS) gene cluster encodes the pituitary-specific hGH-N gene and four highly related genes (hGH-V, hCS-A, hCS-B, and hCS-L) that are expressed only in the placenta. When the hGH-N or hCS-A gene, together with all previously identified cis-acting regulatory sequences, was integrated into the mouse genome, it was expressed only sporadically and at low levels in the transgenic target organs. DNase I mapping of chromatin from expressing and nonexpressing cell types was used to identify a pituitary-specific set of DNase I-hypersensitive sites (HS) and a set of HS common to both the pituitary and placenta, centered approximately 15 and 30 kb 5' of hGH-N, respectively. When contained on a cosmid insert in their native genomic configuration, these HS consistently directed high-level, pituitary-specific expression of hGH-N in transgenic mice and appeared to define a locus control region required for hGH-N expression. Individually, each set of HS was able to mediate position-independent hGH-N expression in the pituitary but demonstrated loss of physiologic control and loss of tissue specificity. The gene-proximal set of HS contained a potent enhancer activity in the pituitary, while the more distal set appeared to function primarily to establish site-of-integration independence. These data indicate that synergistic interactions among multiple elements are required to restrict hGH-N transcription to the pituitary and generate appropriate levels of expression. In addition, these results suggest a role for both shared and unique regulatory sequences in locus control region-mediated expression of the hGH/hCS gene cluster in the pituitary and possibly the placenta.

Adenoma↗
PubMed1994

The LIM/double zinc-finger motif functions as a protein dimerization domain.

Protein-protein interactions resulting in dimerization and heterodimerization are of central importance in the control of gene expression and cell function. Proteins that share the 52-residue LIM/double zinc-finger domain are involved in a wide range of developmental and cellular controls. Some of these functions have been hypothesized to involve protein dimerization. In the present report we demonstrate, using both in vitro and cell-based studies, that a representative LIM protein, human cysteine-rich protein (hCRP), can efficiently homodimerize. The dimerization ability of hCRP is mapped to the LIM domains, can be transferred to an unrelated protein by fusion of a single minimal LIM/double zinc-finger segment, occurs in the absence as well as the presence of DNA, and appears to depend on coordination of two zinc atoms in the finger doublet. These observations support a specific role for protein dimerization in the function of proteins containing the LIM/double zinc-finger domain and expand the general spectrum of potential interactions mediated by zinc-finger motifs.

Amino Acid Sequence↗
PubMed1994

Complex alternative splicing partially inactivates the human chorionic somatomammotropin-like (hCS-L) gene.

The human growth hormone/human chorionic somatomammotropin (hGH/hCS) gene cluster contains five genes: hGH-N, hGH-V, hCS-A, hCS-B, and hCS-L. The expression of the first four genes has been well documented. In contrast, the hCS-L gene has been considered a pseudogene inactivated by loss of the normal intron 2 splice donor site. Previously our laboratory has shown that hCS-L transcripts are present in human placenta and that their levels are induced during the second trimester. More detailed studies of hCS-L transcript processing and mRNA structure are hindered by overwhelming levels of the structurally similar hCS-A and hCS-B transcripts in the placenta. To circumvent this problem, we have established stably transfected cell lines selectively expressing the hCS-L gene. Analysis of hCS-L mRNA from these cell lines demonstrates at least five major alternative splicing pathways, four of which could be confirmed qualitatively by parallel analysis of placental RNA. This analysis reveals an unexpectedly high frequency of exon 2 skipping (73%) as well as utilization of three competing exon 3 splice acceptor sites. Since exon 2 encodes the signal peptide, the majority of hCS-L transcripts are unable to express a secreted protein. Three of the defined hCS-L mRNAs contain an extended open reading frame similar to that present in the functional GH and CS genes. All three of these hCS-L transcripts are of minor abundance and only two, hCS-L(L) and hCS-L(L'), contain exon 2. In vitro translation and signal peptide processing of hCS-L(L) mRNA yields a 20-kDa hCS-L isoform in vitro. These data confirm the placental expression of the hCS-L gene, demonstrate surprising complexity in the splicing of its transcripts, predict that the majority of processed hCS-L mRNAs are nonfunctional, and identify specific, low abundance mRNAs that may encode novel gestational hormones.

Alternative Splicing↗
PubMed1994

Alternative splice site selection in the human growth hormone gene transcript and synthesis of the 20 kDa isoform: role of higher order transcript structure.

Expression of the human growth hormone (hGH) gene in somatotrophs of the anterior pituitary gland results in the synthesis and secretion of a major 22 kDa and a minor 20 kDa GH isohormone. The expression of these two proteins reflects the alternative utilization of a major (B) and a minor (B') splice acceptor site in exon 3 of the hGH-N transcript. By comparing the structure and splicing patterns of the hGH-N gene transcript with that of the structurally related, placentally expressed, hGH-V gene transcript, which uses only the major (B) exon 3 splice acceptor, it has been possible to define the cis-acting elements in exon 3 that are critical for activation of the B' splice acceptor. The present paper demonstrates that, in addition to the importance of sequences in the immediate proximity of the two alternative splice acceptor sites, additional more remote sequences in the transcript also contribute to this alternative splice site selection. The data further suggest that these more distal sequences do not act individually, but interact so that the net level of alternative splicing in exon 3 is dictated by the overall higher order structure of the hGH-N transcript.

Alternative Splicing↗
PubMed1993

Functional characterization of the alternatively spliced, placental human growth hormone receptor.

The human growth hormone family of peptide hormones is encoded by five genes, pituitary growth hormone (hGH-N), and four placentally expressed genes, growth hormone variant (hGH-V), chorionic somatomammotropin A and B (hCS-A, hCS-B), and prolactin (hPrl). As part of an effort to define the local effects of the placentally expressed members of the GH/Prl family of hormones on the placenta, we have identified an isoform (hGHRd3) of the growth hormone receptor expressed in the placental villi. hGHRd3 mRNA differs from the liver GHR mRNA by the deletion of a 66-base pair segment encoding exon 3. In this study we show that hGHRd3 mRNA encodes a stable and functional receptor. hGHRd3 mRNA is efficiently translated and processed in a rabbit reticulocyte lysate translation system as well as in an in vivo Xenopus laevis oocyte expression system. In Xenopus oocytes hGHRd3 is stably integrated into the cell membrane and binds and internalizes ligand as efficiently as hGHR. hGHRd3 binds all three of the placentally expressed members of the GH/Prl gene family (hGH-V, hCS, and Prl) as well as both the 22 and 20 kDa isoforms of the pituitary hGH-N. The results of the present study strongly support the expression of a functional hGHRd3 isoreceptor in the placenta which may serve in autocrine, paracrine, and/or endocrine activation.

Animals↗
PubMed1992

Identification of the sterol- and actin-binding domains of plasma vitamin D binding protein (Gc-globulin).

The mammalian plasma vitamin D binding protein (DBP), or Gc-globulin, is recognized to have at least two functional properties: sterol binding and G-actin sequestration. Affinity labeling of the sterol binding site with the radioactive electrophilic ligand, 3 beta-(bromoacetoxy)-25-hydroxycholecalciferol, followed by limited proteolysis, permitted the isolation and identification of three overlapping peptides in the amino terminus of the molecule. When G-actin affinity chromatography was applied to other proteolytic fragments, two fragments from the carboxy terminus of the molecule were isolated and identified. Another, large, tryptic fragment displayed both sterol- and actin-binding properties. The amino-terminal assignment of the sterol-binding domain was confirmed by demonstrating sterol-specific binding by an in vitro transcribed and translated product of a mutated rat DBP cDNA encoding a protein truncated in its carboxy terminus. The sterol-binding domain was localized to the region between the first-amino-terminal disulfide bond, and the actin-binding domain was found between residues 350 and 403. A high degree of sequence conservation in these regions was found among human, rat, and mouse DBP's. These functional domain assignments confirm the apparent independence of these two binding activities and help to explain the observed triprotein complex of DBP-actin-DNase I and the competition between DBP and profilin for G-actin binding. Our findings should facilitate more precise delineation of the binding domains by site-directed mutagenesis experiments.

Actins↗
PubMed1992

A native RNA secondary structure controls alternative splice-site selection and generates two human growth hormone isoforms.

Consensus sequences at the splice donor, splice acceptor, and lariat branch point regions are necessary but insufficient determinants of splice-site selection in nuclear precursor mRNAs. Sequences outside of these regions can have a significant effect on the utilization of splice sites. Although the mode of action of such sequences is undefined in most cases, higher order RNA structures have been suggested as a potential contributor to splice-site selection. During a detailed analysis of the splicing patterns of the human growth hormone transcript, we located 2 bases in the vicinity of the exon 3 major splice-acceptor site (B) which facilitate the utilization of a competing downstream acceptor (B'). The effects of a series of site-specific mutations on the splicing pattern demonstrate that these 2 bases function by stabilizing a specific stem-loop structure in the native transcript. This defined secondary structure selectively encompasses the upstream B splice-acceptor site together with its lariat branch point region. Increasing the predicted stability of this stem by point mutations results in a corresponding shift in splicing towards the alternative B' splice-acceptor site. These results indicate that a specific secondary structure within the native human growth hormone transcript controls the relative utilization of two competing splice-acceptor sites with the consequent generation of two functionally distinct hormone isoforms.

Animals↗
PubMed1992

Developmental control and alternative splicing of the placentally expressed transcripts from the human growth hormone gene cluster.

Four of the five genes in the human growth hormone gene cluster are expressed in the villous layer of the placenta. We report that the expression of these genes, hCS-A, hCS-B, hCS-L, and hGH-V, are coordinately induced during fetal development, increasing between 12 and 20 weeks of gestation and then plateauing through term. Within the context of this coordinate activation, these genes are expressed at widely different levels and are alternatively spliced in different patterns. There is a developmentally regulated switch in the relative expression of the two chorionic somatomammotropin genes, hCS-A and hCS-B. Starting from approximately equal levels at 8 weeks of gestation, hCS-A is expressed 5-fold more abundantly than hCS-B by term. The proportion of alternatively spliced hGH-V transcripts that retain intron 4 is also developmentally regulated, increasing 3-fold during gestation to 15% at term. A small percentage of hCS transcripts stably retain intron 4 through gestation, the majority derived from the hCS-A gene. hCS-L transcripts undergo two distinct, developmentally stable, splicing pathways between exons 2 and 3. These result from the absence of the normal splice-donor site in intron 2 and the activation of two cryptic splice-acceptor sites. Despite high levels of sequence identity, the four placentally expressed genes in the growth hormone cluster generate a complex set of mRNAs based on alternative splicing and developmental regulation during gestation.

Amino Acid Sequence↗
PubMed1992

Human cysteine-rich protein. A member of the LIM/double-finger family displaying coordinate serum induction with c-myc.

We previously reported the structure of the placentally derived human cysteine-rich (h crp) cDNA and demonstrated that it encodes a highly conserved and widely distributed zinc finger-like protein. We now report that the expression of both the mouse and human crp genes is induced as a primary response to serum in quiescent Balb/c 3T3 cells and in human fibroblasts. The profile of this primary response is remarkably parallel to that of c-myc in the Balb/c 3T3 cell line. The structure of the 23.2-kilobase h crp gene demonstrates that it is a member of a gene superfamily encoding proteins sharing a highly characteristic 52-amino acid "LIM/double-finger" motif. The evolutionarily conserved structure of cysteine-rich protein, its structural similarity to a number of developmentally critical proteins, its distinctive tissue distribution, and its primary response to early events in the cell cycle suggest that crp plays an important role in cell function.

3T3 Cells↗
PubMed1992

Analysis of the human cysteine-rich protein gene (CSRP), assignment to chromosome 1q24-1q32, and identification of an associated MspI polymorphism.

The human cysteine-rich protein (hCRP) is encoded by a highly conserved and widely expressed serum-inducible immediate early response gene. hCRP contains two copies of the "LIM/double zinc-finger" motif. Using a characterized hCRP cDNA probe, we demonstrate that the human CRP gene (CSRP) is present in a single copy and that both mouse and human genomes contain one or more CRP-related genes detected by hybridization at low stringency. Using a panel of human x rodent somatic cell hybrids, the hCRP locus is assigned to chromosome 1. In situ hybridization of 3H-labeled CRP cDNA to human metaphase chromosomes confirms this assignment and permits regional localization to bands 1q24-1q32. A common MspI polymorphism is identified and mapped to intron 4 of the hCRP gene. The chromosomal localization and restriction site polymorphism should prove useful in future studies of the function of this gene.

Animals↗
PubMed1992

Expression of a human growth hormone (hGH) receptor isoform is predicted by tissue-specific alternative splicing of exon 3 of the hGH receptor gene transcript.

Four members of the GH gene family, human (h) GH-V, human chorionic somatomammotropin-A (hCS-A), hCS-B, and hCS-L, are expressed in the human placenta. In attempting to define the role of these hormones in placental development, we have structurally characterized the human placental GH receptor (GHR) mRNA. Human GHR cDNAs were cloned from a human placental cDNA library. Clone pGHR-P1 encompasses the entire hGHR-coding region and is identical to the previously reported liver hGHR cDNA, except for a precise deletion of the sequences corresponding to exon 3. This mRNA with an exon 3 deletion is the sole form of the hGHR mRNAs in the placental villi. A reverse transcription/polymerase chain reaction assay was used to further characterize GHR mRNA expression. Human GHR mRNA was detected in all placental tissues as well as in a wide spectrum of other tissues and cell lines. The distribution of the exon 3-retaining and exon 3-excluding isoforms of the hGHR mRNA shows distinct tissue specificity. Some tissues and cell lines contain only one of the two forms, and some contain a mixed population. Since exon 3 encodes a segment in the extracellular domain of the receptor, its alternative inclusion or exclusion may mediate critical alterations in hormone binding and physiological function.

Amino Acid Sequence↗
PubMed1991

The rat vitamin D binding protein (Gc-globulin) gene. Structural analysis, functional and evolutionary correlations.

The complete rat vitamin D binding protein (DBP) gene has been cloned and characterized. Genomic mapping suggests that there is only one copy of this gene in the haploid genome. The gene spans 35 kilobase pairs and contains 13 exons. All exons, exon/intron borders, and 2196 base pairs of 5'-flanking region have been sequenced. The transcription cap site, determined by primer extension analysis, is 62 base pairs upstream from the start of translation and predicts that an unusual TGTAAA motif may serve as a surrogate TATA. The promoter region contains about 50% nucleotide sequence similarity to the corresponding region of the partially characterized human DBP gene and is uniquely interrupted by a repetitive element. Although lacking in overall sequence similarity to the albumin (ALB) and alpha-fetoprotein (AFP) genes, the 5'-flanking region of the DBP gene contains a number of conserved segments which may correspond to critical proximal promoter elements in this gene family. The location of the introns in the coding region of the DBP gene is highly conserved when compared with the ALB and AFP genes. Detailed comparison of exon size and content confirms the previous prediction that the smaller size of the DBP protein results from loss of internal exons 12 and 13 from the DBP progenitor gene during its evolutionary divergence from ALB and AFP.

Amino Acid Sequence↗
PubMed1991

Molecular characterization of a major nephritogenic domain in the autoantigen of anti-tubular basement membrane disease.

Anti-tubular basement membrane (alpha TBM) disease is a form of primary interstitial nephritis mediated by autoimmune T cells and alpha TBM antibodies. In mice and humans the nephritogenic immune response is directed to a glycoprotein (3M-1) found along the proximal tubule of the kidney. We have isolated cDNAs from an expression library that encodes for the common framework domain of the 3M-1 antigen. This common domain was once related evolutionarily to a family of intermediate filament-associated proteins. Northern hybridization revealed that all isoforms of 3M-1 range between 1700 and 1900 base pairs and in situ hybridization studies indicate that transcripts are found in tubular epithelium. Candidate peptide fragments were deduced and synthesized from the sequence encoding this common framework domain, and one of the peptide residues was able to bind a monoclonal 3M-1-reactive alpha TBM antibody, stimulate the growth of 3M-1-reactive helper T cells, and induce nephritogenic effector T cells capable of producing interstitial nephritis. Our results indicate that a unique, immunodominant region of the 3M-1 antigen is an informative participant in the emergence of autoimmune injury to certain basement membranes.

Amino Acid Sequence↗
PubMed1991

Vitamin D binding protein: genomic structure, functional domains, and mRNA expression in tissues.

The vitamin D binding protein (DBP), alternatively known as Gc-globulin, is a member of the albumin (ALB) and alpha-fetoprotein (AFP) gene family. The rat DBP gene is expressed at high levels in liver and at moderate levels in kidney, testis, abdominal fat, and yolk sac. Very low levels of DBP as well as ALB and AFP transcripts can be detected in all other tissues studied by the reverse transcriptase/polymerase chain reaction technique. During development, liver DBP gene transcripts are detectable at 14 days of gestation and levels rise gradually until adulthood in parallel with ALB. DBP present on the surface of U937 monocyte-derived cells is acquired from serum, suggesting cell surface binding sites for DBP. The rat DBP gene has been cloned and characterized. It spans 35 kb and contains 13 exons and 12 introns. The DBP gene contains two fewer exons than the ALB or AFP genes, accounting for the shortest size of its mRNA and protein product. Its 5'-flanking region contains a high degree of structural similarity to both ALB and AFP promoters.

Amino Acid Sequence↗
PubMed1991

Human growth hormone-variant is a biologically active somatogen and lactogen.

The human GH-variant (hGH-V) gene, a member of the GH-PRL gene family, is expressed by the placenta during the second and third trimesters of gestation. The secreted hGH-V protein differs from pituitary GH (hGH-N) by only 13 amino acids. We have previously demonstrated that hGH-V can bind to both somatogen and lactogen cell surface receptors in vitro, but that the ratio of its somatogen to lactogen receptor-binding affinities is substantially higher than that of hGH-N. We now characterize the somatogen and lactogen bioactivities of hGH-V and contrast them to the bioactivity of hGH-N. Somatogen bioactivity was assayed by stimulation of weight gain in hypophysectomized rats, and lactogen bioactivity was assayed by the mitogenic response of the Nb2 lymphoma cell line. While the average increase in rat body weight in response to a fixed concentration of hormone was comparable using either hGH-V or hGH-N, the mitotic response of the lactogen-inducible Nb2 cells was significantly less for hGH-V. The comparable somatogen, but lower lactogen, bioactivity of hGH-V relative to hGH-N parallels the previously reported receptor binding profiles of the two hormones and suggests that hGH-V has the potential to perform a unique role during human gestation.

Animals↗