A MspI polymorphism at the ovine thyroxine binding globulin locus (TBG).
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Animals↗
Biomedical subjects
D F Hill
Publications and source records attributed to D F Hill.
Sheep linkage mapping: restriction fragment length polymorphism detection with heterologous cDNA probes.
A selection of cattle, human and sheep cDNA probes were screened against sheep genomic DNA, cut with 10 different restriction enzymes, to assess the usefulness of these probes for restriction fragment length polymorphism (RFLP) linkage studies in sheep. Two-thirds of the cattle cDNA probes showed moderate to strong homology with sheep DNA samples, compared with less than half of the human cDNA probes at the final washing stringency chosen for the experiments. The set of probes tested detected a useful frequency of RFLPs. Fifty-seven per cent of probes showing moderate to strong homology identified RFLPs with one or more restriction enzymes. Restriction enzymes that detected RFLPs most frequently in sheep were TaqI and MspI. The results show that sheep and cattle cDNA probes, including candidate genes for production traits, identified a high frequency of RFLPs suitable for genetic mapping in sheep.
Animals↗
Expression of Escherichia coli beta-galactosidase in Mycobacterium bovis BCG using an expression system isolated from Mycobacterium paratuberculosis which induced humoral and cellular immune responses.
A promoter sequence, PAN, was isolated from Mycobacterium paratuberculosis and characterized. This promoter lies adjacent to, and outside, the 3' end of an IS900 insertion element. IS900 contains an open reading frame, ORF2, on the complementary strand which codes for the putative transposase of this insertion sequence. A DNA fragment containing PAN and part of ORF2 was fused to the lacZ gene and inserted into the replicative shuttle vector pRR3. Mycobacterium smegmatis and Mycobacterium bovis BCG (BCG) transformed with this plasmid exhibited beta-galactosidase activity. However, lacZ was only expressed in Escherichia coli under the control of PAN, when ORF2 was deleted. Immunization of mice with the recombinant M. bovis BCG expressing lacZ resulted in the induction of a high humoral and cellular response directed against beta-galactosidase. The PAN-ORF2 expression system may prove to be particularly useful for cloning and expression of heterologous genes in the BCG vaccine strain.
Amino Acid Sequence↗
Ovine follistatin: characterization of cDNA and expression in sheep ovary during the luteal phase of the oestrous cycle.
We have isolated ovine follistatin cDNA from an ovarian follicle cDNA library and determined its sequence. The deduced amino acid sequence of the ovine follistatin precursor is highly homologous (greater than 97%) to the porcine, human and rat follistatins. Northern analysis was used to characterize follistatin gene expression in ovaries of adult ewes, collected from days 11 to 13 of the oestrous cycle. Two major (about 2.7 kb and 1.5 kb) and one minor (about 0.5 kb) transcripts were detected in polyadenylated RNA extracted from ovarian follicles and corpora lutea. The degree of expression of the transcripts varied in the two ovarian compartments, with the 2.7 kb species predominating in the follicles and the 1.5 kb species being more abundant in the corpora lutea. No transcripts were detected in stromal tissue containing preantral follicles of less than 1 mm.
Amino Acid Sequence↗
Transforming growth factor alpha contributes to the mechanism by which hypothalamic injury induces precocious puberty.
It has long been known that lesions of the hypothalamus lead to female sexual precocity. While an increased production of luteinizing hormone-releasing hormone (LHRH), the neurohormone that controls sexual development, appears to mediate the advancement of puberty induced by these lesions, little is known about the mechanism(s) by which hypothalamic injury activates LHRH secretion. Since brain lesions result in accumulation of neurotrophic/mitogenic activities in the injured area, we tested the hypothesis that transforming growth factor alpha (TGF-alpha), a mitogenic polypeptide recently shown to stimulate LHRH release, is produced in response to hypothalamic injury and mediates the effect of the lesion on puberty. Radiofrequency lesions of the preoptic area-anterior hypothalamic area (POA-AHA) of 22-day-old female rats resulted in precocious puberty within 7 days after the operation. RNA blot hybridization revealed that lesion-induced puberty was preceded by an increase in TGF-alpha mRNA levels in the POA-AHA. Epidermal growth factor (EGF) mRNA was undetectable in both intact and lesioned hypothalami. TGF-alpha mRNA levels, quantitated by RNase protection assays, were 3.5-fold greater in lesioned animals approaching puberty than in age-matched controls. Immunohistochemical studies, utilizing single- and double-staining procedures, demonstrated the presence of TGF-alpha precursor-like immunoreactivity in reactive astrocytes surrounding the lesion site. Hybridization histochemistry showed increased TGF-alpha mRNA expression in cells of the same area, further implicating reactive astrocytes as a site of TGF-alpha synthesis. The actions of TGF-alpha are mediated by its interaction with EGF receptors. Continuous infusion of RG-50864, an inhibitor of EGF receptor kinase activity, at the site of injury prevented the advancement of puberty induced by the lesion. These results suggest that TGF-alpha acting via EGF-like receptors contributes to the acceleration of puberty induced by anterior hypothalamic lesions. They also indicate that activation of TGF-alpha gene expression in glial cells is a component of the hypothalamic response to injury.
Animals↗
The putative single-stranded DNA-binding protein of the filamentous bacteriophage, Ifl. Amino acid sequence of the protein and structure of the gene.
The protein product corresponding to the gene located in the region of the coliphage Ifl genome shown to contain the code for the single-stranded DNA (ssDNA)-binding proteins of all filamentous phages so far studied has been isolated from infected bacterial cells and its amino acid sequence determined. The mature protein contains 95 amino acids (calculated molecular mass 10553 Da). Its sequence corresponds to that predicted from the DNA sequence but lacks the initiating methionine residue. Although there is little direct sequence homology between the phage Ifl protein and the ssDNA-binding proteins of the other filamentous phages that have been studied, computer-based comparisons of various physical and structural parameters showed that the phage Ifl protein contains a domain that is closely related to domains in the coliphage T4 gene 32 protein and the Pseudomonas phage Pfl ssDNA-binding protein and suggest that the Ifl protein does have a ssDNA-binding function although we were unable to show this directly.
Amino Acid Sequence↗
DNA sequence of the filamentous bacteriophage Pf1.
The genome of the class II filamentous bacteriophage Pf1 has been sequenced by a combination of the chain termination and chemical degradation methods. It consists of 7349 nucleotides in a closed, circular loop of single-stranded DNA. The size and position of its open reading frames (ORFs) in general resemble those of other filamentous bacteriophage genomes. The size and position of the spaces between the ORFs have not been conserved, however, and six short reading frames (2 of which overlap) occupy a region corresponding to that filled by genes 2 and 10 in the Ff genome. Most of the ORFs are preceded by sequences resembling ribosome binding sites from the phage's host. Pseudomonas aeruginosa, that appear to differ somewhat from their counterparts in Escherichia coli. A search for sequences related to known pseudomonad promoters suggests that the promoters in this bacteriophage may well be ntr-dependent, with the two strongest preceding the gene for the major coat protein (gene 8) and another ORF (430). Gene 8 is followed by a sequence with the properties of a rho-independent terminator of transcription, like that at the same position in the genome of Ff. The Pf1 genome contains no collection of potential stem-and-loop structures corresponding to those that initiate replication of Ff DNA and assembly of the Ff virion, although isolated structures of this kind are present. The available evidence suggests that at least 13 of the 14 major ORFs are expressed. Overall, the organization of the Pf1 genome differs from that of the other class II filamentous phage whose genome has been sequenced, Pf3, as much as it does from that of the class I phages Ff and IKe.
Amino Acid Sequence↗
The genes encoding nerve growth factor and its receptor are expressed in the developing female rat hypothalamus.
Nerve growth factor (NGF), its messenger RNA (mRNA) and its receptor protein and mRNA are found in several brain regions. Little attention has been given to the possibility that the hypothalamus, which controls the endocrine system, may produce NGF and/or express NGF receptors. This would indicate the existence in this brain area of neuronal populations which are either target for NGF-responsive cells or sensitive to NGF, respectively. Blot hybridization of polyadenylated (poly(A)+) RNA to a mouse NGF cRNA probe revealed the presence of NGF mRNA in both the suprachiasmatic region (henceforth, called preoptic area [POA]) and medial basal hypothalamus (MBH) of developing female rats. The mRNA was already detectable on fetal day 18 and reached maximal levels around postnatal day 3 (MBH) and 12 (POA), declining thereafter. This pattern was temporally different than that of areas known to produce NGF, i.e., the neocortex (Cc) and hippocampus (Hc). In agreement with previous reports, NGF mRNA levels in these areas were negligible before birth, became maximal between the second and third week of postnatal life and decreased moderately thereafter. The concentration of NGF protein, measured by a two-site enzyme immunoassay, was 3 times higher in the POA than in the MBH at an infantile age (day 12), increasing 2-fold in the POA of juvenile animals (day 28) but not in the MBH. This developmental pattern was similar to that seen in the Hc, though the NGF concentrations were significantly lower in the POA.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Repeat sequences from complex ds DNA viruses can be used as minisatellite probes for DNA fingerprinting.
In a search for new fingerprinting probes for use with sheep, repeat sequences derived from five poxviruses, an iridovirus and a baculovirus were screened against DNA from sheep pedigrees. Probes constructed from portions of the parapox viruses, orf virus and papular stomatitis virus and the baculovirus from the alfalfa looper, Autographa californica, nuclear polyhedrosis virus all gave fingerprint patterns. Probes from three other poxviruses and an iridovirus did not give useful banding patterns.
Animals↗
A MspI restriction fragment length polymorphism at the ovine locus for neurotensin.
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Alleles↗
A RsaI restriction fragment length polymorphism at the ovine locus for gamma-interferon.
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Alleles↗
Nerve growth factor receptors in the peripubertal rat ovary.
We previously demonstrated that the immature rat ovary synthesizes nerve growth factor (NGF), and that interference of NGF actions by immunoneutralization during neonatal life prevents development of the ovarian sympathetic innervation and delays follicular maturation. Since the actions of NGF are exerted via binding to specific cell surface receptors, the present study was undertaken to define and characterize the presence of NGF receptors (NGFrec) in the developing rat ovary. NGF interacts with two classes of NGFrec. The most abundant is a low affinity form expressed in the central nervous system and peripheral tissues. This receptor is encoded by a single 3.8-kilobase mRNA species. Cross-linking of [125I]NGF to ovarian membranes followed by immunoprecipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and autoradiography showed the presence of a approximately 90-kilodalton molecular species which corresponds in size to the predominant NGF receptor species cross-linked to its ligand. While ovarian NGFrec may be of neuronal origin and reach the gland exclusively by anterograde axonal transport, RNA blot hybridization demonstrated that the ovary expresses the NGFrec mRNA species that encodes the low affinity NGF receptor and, thus, implicated the ovary itself as a site of NGFrec synthesis. NGFrec mRNA levels decreased abruptly after the first ovulation, suggesting that NGFrec may be synthesized in growing follicles and that this capacity is lost after follicular rupture and luteinization.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
A MspI restriction fragment length polymorphism at the ovine locus for 2',3'-cyclic-nucleotide 3'-phosphohydrolase.
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2',3'-Cyclic-Nucleotide Phosphodiesterases↗
Sequence analysis of the inverted terminal repetition in the genome of the parapoxvirus, orf virus.
Two BamHI fragments from the right-hand terminal region of the orf virus genome have been sequenced. The bulk of the inverted terminal repetition (ITR) sequence is contained within these fragments and makes up 3388 bp of the 4425-bp sequence reported. The overall base composition of the larger sequence is 59.4% G + C and of the ITR, 60.2% G + C. An extremely G/C-rich (83.2%) block of sequence was found spanning the ITR/unique sequence junction. The bulk of the ITR could be divided into three blocks of directly repeated sequences. One block begins about 250 nucleotides from the terminus and is a direct repeat 15 bp long, repeated 14 times. The other blocks contain seven sequence sets ranging from 16 to 36 nucleotides which are repeated 2 to 4 times, interspersed with one another, interrelated in sequence, and sometimes separated by unique sequence. Eight open reading frames (ORFs), each with the potential to code for polypeptides of 50 residues or more, were identified. Three were found within the ITR, four spanned the ITR/unique sequence junction and one was found outside the ITR. A search for putative poxvirus transcriptional control signals indicated that three of the eight ORFs are likely to be transcribed early, all in the same direction toward the right end of the genome. Sequences of the type T(A)3-5T were found only twice in the sequence and only one preceded an ORF.
Amino Acid Sequence↗
Involvement of transforming growth factor alpha in the release of luteinizing hormone-releasing hormone from the developing female hypothalamus.
Little is known about the presence of trophic factors in the hypothalamus and the role they may play in regulating the functional development of hypothalamic neurons. We have investigated the ability of epidermal growth factor (EGF) and transforming growth factor alpha (TGF-alpha) to affect the release of luteinizing hormone-releasing hormone (LHRH), the neuropeptide that controls reproductive development. We have also determined whether the genes encoding EGF and TGF-alpha are expressed in the prepubertal female hypothalamus. Northern blot analysis of poly(A)+ RNA utilizing a single-stranded EGF cDNA probe failed to reveal the presence of EGF mRNA in either the hypothalamus or the cerebral cortex at any age studied (fetal day 18 to postnatal day 36). In contrast, both a complementary RNA probe and a double-stranded TGF-alpha cDNA recognized in these regions a 4.5-kilobase (kb) mRNA species identical to TGF-alpha mRNA. The abundance of TGF-alpha mRNA was 3-4 times greater in the hypothalamus than in the cerebral cortex. Both EGF and TGF-alpha (2-100 ng/ml) elicited a dose-related increase in LHRH release from the median eminence of juvenile rats in vitro. They also enhanced prostaglandin E2 (PGE2) release. The transforming growth factors TGF-beta 1 and -beta 2 were ineffective. Only a high dose of basic fibroblast growth factor was able to increase LHRH and PGE2 release. Blockade of the EGF receptor transduction mechanism with RG 50864, a selective inhibitor of EGF receptor tyrosine kinase activity, prevented the effect of both EGF and TGF-alpha on LHRH and PGE2 release but failed to inhibit the stimulatory effect of PGE2 on LHRH release. Inhibition of prostaglandin synthesis abolished the effect of TGF-alpha on LHRH, indicating that PGE2 mediates TGF-alpha-induced LHRH release. The results indicate that the effect of EGF and TGF-alpha on LHRH release is mediated by the EGF/TGF-alpha receptor and suggest that TGF-alpha rather than EGF may be the physiological ligand for this interaction. Since in the central nervous system most EGF/TGF-alpha receptors are located on glial cells, the results also raise the possibility that--at the median eminence--TGF-alpha action may involve a glial-neuronal interaction, a mechanism by which the trophic factor first stimulates PGE2 release from glial cells, and then PGE2 elicits LHRH from the neuronal terminals.
Aging↗
The gene encoding nerve growth factor is expressed in the immature rat ovary: effect of denervation and hormonal treatment.
The rat ovary is innervated by sympathetic nerve fibers. Since the development and survival of peripheral sympathetic neurons innervating nonreproductive organs have been shown to depend on the production of nerve growth factor (NGF) by the innervated tissues, the present experiments were undertaken to determine if the immature rat ovary has the capability of synthesizing NGF. Blot hybridization of ovarian polyadenylated RNA (A+-RNA) to a NGF cRNA probe revealed the presence of a 1.3- to 1.4-kilobase (kb) mRNA species similar to mature NGF mRNA detected in mouse submaxillary gland, a source rich in NGF. Quantitation of NGF protein by a sensitive and specific two-site enzyme immunoassay demonstrated the presence of NGF in juvenile ovaries at levels comparable to those found in other sympathetically innervated tissues. Neither denervation of the ovary nor treatment with gonadotropins (hCG and FSH) or somatomammotropins (PRL and GH) affected the levels of NGF mRNA. However, denervation significantly increased NGF levels, suggesting that, as in other target tissues, denervation prevents the retrograde transport of NGF by the sympathetic terminals and leads to accumulation of the protein at its site of production. It is concluded that 1) the developing ovary is able to both transcribe the NGF gene and translate its mRNA into NGF protein; and 2) the NGF content in the ovary is regulated by its innervation. The results provide the biochemical basis for the concept, elaborated in the companion paper, that NGF through its trophic actions on ovarian sympathetic neurons contributes to the regulation of ovarian development and, hence, to the acquisition of female reproductive capacity.
Animals↗
An autosomal transcript in skeletal muscle with homology to dystrophin.
The Duchenne muscular dystrophy (DMD) gene has been localized to chromosome Xp21 and codes for a 14-kilobase (kb) transcript and a protein called dystrophin, of relative molecular mass 427,000. Dystrophin is associated with the cytoplasmic face of muscle fibre membranes and its C-terminal domain is thought to mediate membrane attachment. Although N-terminal and central domain structures share common features with other cytoskeletal components, no significant sequence similarity between the C-terminal region of dystrophin and other previously characterized proteins has been described. Here we report that fragments from the C-terminal domain of the DMD complementary DNA detect a closely related sequence which exhibits nucleic-acid and predicted amino-acid identities with dystrophin of approximately 65 and 80%, respectively. The dystrophin-related sequence identifies a 13-kb transcript in human fetal muscle and maps to chromosome 6. Thus, dystrophin may be a member of a family of functionally related large structural proteins in muscle.
Base Sequence↗
The isolation and characterization of a cDNA clone encoding Lupinus angustifolius root nodule glutamine synthetase.
Glutamine synthetase, purified from Lupinus angustifolius legume nodules, was carboxymethylated and succinylated prior to chemical or enzymatic cleavage. Peptides were purified and sequenced. An oligonucleotide probe was constructed for the sequence MPGQW. This probe was used to identify a glutamine synthetase cDNA clone, pGS5, from a lupin nodule cDNA library constructed in pBR322. pGS5 was sequenced (1043 bp) and computer-assisted homology searching revealed a high degree of conservation between this lupin partial cDNA clone and other plant glutamine synthetases at both the amino acid (greater than 90%) and nucleotide (greater than 80%) level. Northern and Southern analyses using pGS5 supported the conclusion that a multigene glutamine synthetase family exists in lupin which is differentially expressed in both an organ-specific and temporal manner. Western and Northern blot analyses indicated the accumulation of a glutamine synthetase specific mRNA species during nodule development corresponded to the appearance of a novel glutamine synthetase polypeptide between 8 and 10 days after rhizobial inoculation.
Adenosine Triphosphate↗