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Biomedical subjects

H M Sang

Publications and source records attributed to H M Sang.

14 recordsLinked to original sources

Disease-resistant genetically modified animals.

Infectious disease adversely affects livestock production and animal welfare, and has impacts upon both human health and public perception of livestock production. The authors argue that the combination of new methodology that enables the efficient production of genetically-modified (GM) animals with exciting new tools to alter gene activity makes the applications of transgenic animals for the benefit of animal (and human health) increasingly likely. This is illustrated through descriptions of specific examples. This technology is likely to have specific application where genetic variation does not exist in a given population or species and where novel genetic improvements can be engineered. These engineered animals would provide valuable models with which to investigate disease progression and evaluate this approach to controlling the disease. The authors propose that the use of GM animals will complement the more traditional tactics to combat disease, and will provide novel intervention strategies that are not possible through the established approaches.

Animals↗

Chronology of events in the first cell cycle of the polyspermic egg of the domestic fowl (Gallus domesticus).

The nuclear population in the polyspermic egg of the domestic hen was examined in whole-mount preparations of the germinal disc. The numbers of nuclei varied in groups of hens from averages of 5.9 to 26, depending on days from insemination. Changes in development from initial formation of pronuclei to the early mitoses of the zygote nucleus were staged according to the position of the egg in the oviduct. The findings substantiated earlier accounts on the timing of the apposition of the parental pronuclei towards the end of the first cell cycle. Additionally, analysis of the spatial distribution of accessory spermatozoal nuclei showed a slight, but significant, dispersal from a clustered arrangement at this time.

Animals↗

Regulation of chicken gonadotropin-releasing hormone-I mRNA in incubating, nest-deprived and laying bantam hens.

Secretion of luteinizing hormone is decreased when hens start to incubate their eggs and is increased after nest deprivation or hatching of the eggs. The purpose of this study was to determine whether decreased luteinizing hormone (LH) secretion during incubation in the domestic hen is associated with a decrease in hypothalamic chicken gonadotropin-releasing hormone-I (cGnRH-I) mRNA or peptide. A semiquantitative competitive PCR assay was developed to measure cGnRH-I mRNA. Hypothalamic mRNA was quantified as the amount of GnRH cDNA obtained by reverse transcription of cGnRH-I mRNA. The amount of hypothalamic cGnRH-I mRNA was significantly higher in laying than in incubating hens (38.7 +/- 10.3 vs. 7.7 +/- 1.6 x 10(-17) mol cDNA, p = 0.01, n = 8). The hypothalamic GnRH peptide content was not significantly different between laying and incubating hens in either the preoptic area (286.9 +/- 24.01 vs. 269.3 +/- 29.3 pg, n = 8) or the basal hypothalamus (1.67 +/- 0.19 vs. 1.54 +/- 0.21 ng, n = 8). Five days after incubating hens were deprived of their eggs, the resulting increase in LH secretion was associated with a significant increase in hypothalamic content of cGnRH-I mRNA (22.8 +/- 2.2 vs. 6.7 +/- 1.7 x 10(-17) mol cDNA, p < 0.001, n = 8). These observations suggest that a decrease in the expression of the cGnRH-I gene is a major factor in maintaining depressed LH secretion in incubating domestic chickens.

Animals↗

Evidence for alternative splicing of the chicken vasoactive intestinal polypeptide gene transcript.

Two forms of chicken vasoactive intestinal polypeptide (VIP) mRNA have been identified by reverse transcription (RT)-PCR and RNase protection assay. The shorter form of chicken VIP mRNA encodes a protein that does not contain an analogue of rat peptide histidine isoleucine (PHI) 1-27 or human peptide histidine methionine 1-27. The larger form encodes both VIP and a chicken analogue of PHI 1-27 in the same protein product. Three VIP cDNAs isolated from a chicken hypothalamic cDNA library were derived from the shorter mRNA. Sequence analysis of the longest clone identified an open reading frame that codes for a 165 amino acid preproVIP protein and contains two polyadenylation signals. In situ hybridisation with an oligonucleotide probe from the VIP cDNA sequence showed that VIP-encoding mRNA occurs in cells in the basal hypothalamus, an area of the brain known to contain VIP neurosecretory neurones. RT-PCR of total RNA from liver, kidney, gut, pancreas, pituitary, cerebellum, forebrain and hypothalamus, using primers derived from the VIP cDNA sequence, showed that the shorter form of VIP mRNA is present in all of these tissues. The sequence of the longer form of VIP mRNA was obtained by sequencing a portion of the VIP gene from genomic DNA. This revealed a potential exon that was not represented in the VIP cDNA clones analysed. RT-PCR with primers from this sequence showed that it was expressed in the gut and hypothalamus. RNase protection assays confirmed the presence of the two forms of mRNA in gut and hypothalamus. The relative proportions of the two mRNA forms were: 97.8% VIP only, 2.2% PHI/VIP in the hypothalamus and 98.5% VIP only, 1.5% PHI/VIP in the gut. In conclusion, chicken VIP mRNA is alternatively spliced. The shortest form, which encodes a preproprotein containing only the VIP peptide, is the most abundant. The longer form of chicken VIP mRNA encodes a preproprotein containing sequences for both VIP and a chicken form of PHI.

Alternative Splicing↗

Effect of active immunization against recombinant-derived chicken prolactin fusion protein on the onset of broodiness and photoinduced egg laying in bantam hens.

The hypothesis that the onset of incubation behaviour (broodiness) in the domestic hen is induced by an increase in prolactin secretion was investigated by actively immunizing bantam hens against recombinant-derived chicken prolactin. A second objective was to establish whether active immunization against prolactin affects photoinduced onset of egg laying and the rate of egg production. The immunogen was a fusion protein (beta gals-prolactin, 23 kDa) produced in Escherichia coli, comprising chicken prolactin (without the nine amino-terminal amino acids) fused to 18 amino acids of E. coli beta-galactosidase. A control immunogen was produced in the same strain of E. coli harbouring the same plasmid vector used to produce beta gals-prolactin minus the prolactin gene sequence. Hens were immunized i.m. with 1 mg of protein containing 0.8-0.9 mg of fusion protein in Freund's incomplete adjuvant at 4-8 week intervals beginning before or after egg laying, which was induced by increasing the daily photoperiod. The beta gals-prolactin immunogen, but not the control immunogen, stimulated the production of antibodies to chicken prolactin. In Expts 1, 2 and 3, hens were placed in floor pens with nest boxes after photostimulation to induce broodiness. In these experiments, immunization with beta gals-prolactin reduced the incidence or delayed the development of broodiness. This effect was more pronounced if immunization was initiated before, rather than after, the onset of egg laying. In Expts 1 and 2 hens were immunized with beta gals-prolactin before photostimulation. The presence of antibodies to prolactin in their blood did not affect photoinduced onset of egg laying.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Transgenesis in chickens.

The application of transgenic technology to domestic poultry offers an alternative means to conventional practice for improvement of this highly productive agricultural species. The hen's reproductive system has unique characteristics which have imposed limitations on the use of established methods for artificial gene transfer. In this article, we review the various strategies that have been adopted to overcome the problem. Target sites for gene insertion include the fertilized ovum, the blastodermal embryo in the unincubated egg, and the primordial germ cells. Notable success in obtaining somatic and germline transformation has been achieved with the use of retroviral vectors to infect the blastodermal embryo. Current attempts to introduce DNA directly into the genome, without resort to pathogen-derived vectors, are discussed.

Animals↗

Characterization of the chicken preprogonadotrophin-releasing hormone-I gene.

Partial cDNA clones for chicken gonadotrophin-releasing hormone (GnRH)-I were isolated by reverse transcription-polymerase chain reaction using total RNA from the hypothalami of domestic chickens. Primers for amplification were based on the nucleotide sequence of the mammalian GnRH genes. These amplified clones were used to screen a genomic library from which a series of overlapping clones was isolated. A 6.3 kb EcoRI fragment containing all the exons and 3.0 kb of the 5' upstream region was sequenced. The exon-intron structure of the gene was found to be of a similar configuration to those of the mammalian and osteichthyes GnRH genes analysed so far. Individual domains of the predicted prepropeptide are similar to those of mammalian GnRH prepropeptides, comprising a 23 amino acid signal peptide, the decapeptide hormone and a Gly-Lys-Arg cleavage site, followed by a 56 amino acid GnRH-associated peptide. The nucleotide sequence coding for the decapeptide hormone translates into the amino sequence for chicken GnRH-I. The prepropeptide has approximately 50% identity with mammalian prepropeptides and 25% identity with the teleost prepropeptides.

Amino Acid Sequence↗

Pituitary prolactin messenger ribonucleic acid levels in incubating and laying hens: effects of manipulating plasma levels of vasoactive intestinal polypeptide.

Pituitary PRL messenger RNA levels in hens, measured by dot-blot hybridization, correlated directly with concentrations of plasma PRL, being 3-fold higher in incubating than in laying birds. Nest deprivation of incubating hens for 24 h caused a rapid decrease in both plasma PRL and pituitary PRL mRNA, which remained depressed thereafter. A single injection of vasoactive intestinal polypeptide (VIP) in laying hens resulted in an increase (P less than 0.05) in pituitary PRL mRNA whereas passive immunoneutralization of VIP in incubating hens resulted in a decrease (P less than 0.001) in pituitary PRL mRNA. The rapid decrease in pituitary PRL mRNA after nest deprivation or passive immunoneutralization of VIP was associated with a significant increase in pituitary PRL content, presumably a consequence of the decreased PRL secretion. In situ hybridization showed PRL mRNA to be localized in the cephalic lobe of the anterior pituitary gland in which most PRL cells, identified immunocytochemically, were found. Northern blotting studies showed that the pituitary gland contains a single 860 base(s) mature PRL mRNA transcript irrespective of physiological state or VIP manipulation. Both in situ and Northern hybridization studies confirmed that the amount of pituitary PRL mRNA was related directly to the concentration of plasma PRL. These observations are consistent with the view that in incubating hens hypothalamic VIP, in addition to acting as a PRL releasing hormone, also plays a major role in the regulation of the amount of PRL mRNA in the anterior pituitary gland.

Animals↗

Molecular cloning and sequence analysis of putative chicken prolactin cDNA.

A cDNA library was prepared from mRNA isolated from anterior pituitary glands of incubating bantam hens, in which prolactin mRNA levels were predicted to be very high. Nine clones, representing abundant mRNA species, were identified and shown to contain homologous sequences. Two clones, of 871 bp and 580 bp, were analysed by DNA sequencing. The shorter clone was found to be a truncated cDNA product but otherwise identical to the longer clone. The 871 bp cDNA, PRL101, contains an open reading frame capable of encoding a polypeptide of 229 amino acids. This putative polypeptide has a high degree of homology to mammalian prolactins (approximately 70%), strongly suggesting that PRL101 encodes chicken preprolactin. The protein was predicted to have a 30 amino acid signal sequence which would be cleaved off to give a mature protein of 199 amino acids. The peptide sequence also had a 26% homology to chicken growth hormone, which is related to prolactin. This similarity confirms the conclusion that PRL101 is a chicken prolactin cDNA clone. An abundant mRNA of approximately 880 b was detected in poly(A)+ RNA from pituitary glands probed with PRL101. Analysis of chicken genomic DNA showed that there is one copy of the prolactin gene in the genome. PRL101 hybridized strongly to genomic DNA from closely related galliforms (quail and turkey) and less strongly to DNA from more distantly related species (duck and ring dove).

Amino Acid Sequence↗

Expression of biologically active recombinant-derived chicken prolactin in Escherichia coli.

The putative chicken prolactin (chPRL) cDNA clone PRL101 was manipulated in vitro and cloned into the Escherichia coli expression vector pKK2332 to produce a plasmid coding for recombinant-derived mature chPRL (R-chPRL). Expression of this manipulated cDNA sequence in E. coli resulted in the production of a 23 kDa protein which cross-reacted with specific chPRL antisera in Western blots. The partially purified protein stimulated ring dove crop sac mucosa to proliferate in a PRL bioassay, demonstrating that the R-chPRL was biologically active. R-chPRL was expressed at a level of approximately 1.5% of total cell protein.

Animals↗

Molecular lesions associated with white gene mutations induced by I-R hybrid dysgenesis in Drosophila melanogaster.

We have identified molecular lesions associated with six mutations, w and w, of the white gene of Drosophila melanogaster. These mutations arose in flies subject to I-R hybrid dysgenesis. Four of the mutations give rise to coloured eyes and are associated with insertions of 5.4-kb elements indistinguishable from the I factor controlling I-R dysgenesis. The insertion associated with w is at a site which, within the resolution of these experiments, is identical to that of two previously studied I factors. This appears to be a hot-spot for I factor insertion. We have compared the sites of these insertions with sequences complementary to white gene mRNA identified by Pirrotta and Bröckl. The hot-spot is in the fourth intron. The insertion carried by w is either within, or just beyond, the last exon. The insertion carried by w is near the junction of the first exon and first intron. The w mutation is a derivative of w. It contains an insertion of I factor DNA within, or immediately adjacent to, the F-like element associated with w, and results in restoration of some eye colour. This insertion is just upstream of the start of the white mRNA. Mutations w and w are deletions removing mRNA coding sequences. Both determine a bleached white phenotype.

Journal Article↗

The molecular basis of I-R hybrid dysgenesis in Drosophila melanogaster: identification, cloning, and properties of the I factor.

We have analyzed two mutations of the white-eye gene, which arose in flies subject to I-R hybrid dysgenesis. These mutations are associated with insertions of apparently identical 5.4 kb sequences, which we have cloned. We believe that these insertions are copies of the I factor controlling I-R hybrid dysgenesis. The I factor is not a member of the copia-like or fold-back classes of transposable elements and has no sequence homology with the P factor that controls P-M dysgenesis. All strains of D. melanogaster contain I-factor sequences. Those present in reactive strains must represent inactive I elements. I elements have a remarkably similar sequence organization in all reactive strains and are located in peri-centromeric regions. Inducer strains appear to contain both I elements, located in peri-centromeric regions, and 10-15 copies of the complete I factor at sites on the chromosome arms.

Animals↗