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M. C. Hanks, R. T. Talbot, and H. M. Sang

ABSTRACT

The putative chicken prolactin (chPRL) cDNA clone PRL101 was manipulated in vitro and cloned into the Escherichia coli expression vector pKK233-2 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.

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M.C. Hanks, J.A. Alonzi, P.J. Sharp, and H.M. Sang

ABSTRACT

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).

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R T Talbot, I C Dunn, P W Wilson, H M Sang, and P J Sharp

ABSTRACT

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.

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I C Dunn, Y Chen, C Hook, P J Sharp, and H M Sang

ABSTRACT

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 63 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.