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Biomedical subjects

P B Hackett

Publications and source records attributed to P B Hackett.

At least 37 records · Page 2Linked to original sources
PubMed1995

Regulation of fish growth hormone transcription.

Regulation of endogenous fish growth hormone transcription was studied using carp pituitaries in vitro. It was demonstrated that thyroid hormone (T3) and 9-cis retinoic acid have increased the steady state levels of growth hormone messenger RNA in pituitary cells, as compared with beta-actin messenger RNA levels. In contrast, estrogen failed to increase growth hormone mRNA levels. The possible involvement of thyroid hormone receptor in pituitary gene expression was demonstrated by in situ localization of both growth hormone mRNA and thyroid hormone receptor mRNA in the pituitaries as early as 4 days after fertilization.

Actins↗
PubMed1995

Development of position-independent expression vectors and their transfer into transgenic fish.

Many genes have been transferred into fish for scientific and aquacultural purposes. We have been developing expression vectors containing regulatory sequences from the carp beta-actin gene enhancer/promoter for expression of genes or cDNAs in transgenic fish. Expression from these vectors varies over a 20-fold range in zebrafish, beginning within 12 hours of fertilization and continuing for at least two weeks. Expression can be found in nearly all tissues. The vectors have the following characteristics: (1) they contain either unique or polycloning restriction endonuclease sites for insertion of any gene or cDNA, and (2) the piscine sequences are flanked by restriction sites for easy removal of plasmid, or nonfish, sequences. We have tested the ability of special sequences, border elements, from other animals to confer position-independent expression of transgenes or enhance integration of transgenic constructs into fish chromosomes. Early results indicate that these elements do not act as enhancers and do not improve integration frequencies. However, both avian and insect border elements are able to confer position-independent expression as judged from expression of CAT genes in F1 generation fish.

Animals↗
PubMed1993

Regulation of expression of transgenes in developing fish.

The transcriptional regulatory elements of the beta-actin gene of carp (Cyprinus carpio) have been examined in zebrafish and goldfish harbouring transgenes. The high sequence conservation of the putative regulatory elements in the beta-actin genes of animals suggested that their function would be conserved, so that transgenic constructs with the same transcriptional control elements would promote similar levels of transgene expression in different species of transgenic animals. To test this assumption, we analysed the temporal expression of a reporter gene under the control of transcriptional control sequences from the carp beta-actin gene in zebrafish (Brachydanio rerio) and goldfish (Carrasius auratus). Our results indicated that, contrary to expectations, combinations of different transcriptional control elements affected the level, duration, and onset of gene expression differently in developing zebrafish and goldfish. The major differences in expression of beta-actin/CAT (chloramphenicol acetyltransferase) constructs in zebrafish and goldfish were: (1) overall expression was almost 100-fold higher in goldfish than in zebrafish embryos, (2) the first intron had an enhancing effect on gene expression in zebrafish but not in goldfish, and (3) the serum-responsive/CArG-containing regulatory element in the proximal promoter was not always required for maximal CAT activity in goldfish, but was required in zebrafish. These results suggest that in the zebrafish, but not in the goldfish, there may be interactions between motifs in the proximal promoter and the first intron which appear to be required for maximal enhancement of transcription.

Actins↗
PubMed1993

Alterations of the three short open reading frames in the Rous sarcoma virus leader RNA modulate viral replication and gene expression.

The Rous sarcoma virus (RSV) leader RNA has three short open reading frames (ORF1 to ORF3) which are conserved in all avian sarcoma-leukosis retroviruses. Effects on virus propagation were determined following three types of alterations in the ORFs: (i) replacement of AUG initiation codons in order to prohibit ORF translation, (ii) alterations of the codon context around the AUG initiation codon to enhance translation of the normally silent ORF3, and (iii) elongation of the ORF coding sequences. Mutagenesis of the AUG codons for ORF1 and ORF2 (AUG1 and AUG2) singly or together delayed the onset of viral replication and cell transformation. In contrast, mutagenesis of AUG3 almost completely suppressed these viral activities. Mutagenesis of ORF3 to enhance its translation inhibited viral propagation. When the mutant ORF3 included an additional frameshift mutation which extended the ORF beyond the initiation site for the gag, gag-pol, and env proteins, host cells were initially transformed but died soon thereafter. Elongation of ORF1 from 7 to 62 codons led to the accumulation of transformation-defective virus with a delayed onset of replication. In contrast, viruses with elongation of ORF1 from 7 to 30 codons, ORF2 from 16 to 48 codons, or ORF3 from 9 to 64 codons, without any alterations in the AUG context, exhibited wild-type phenotypes. These results are consistent with a model that translation of the ORFs is necessary to facilitate virus production.

Animals↗
PubMed1993

Effects of the open reading frames in the Rous sarcoma virus leader RNA on translation.

Three short open reading frames (ORFs) reside in the 5' leader of Rous sarcoma virus (RSV) and are conserved in all avian sarcoma-leukosis retroviruses. Both extensions of the lengths of the ORFs and alterations in their initiation codons affect viral replication and gene expression. To determine whether the effects on viral replication were due to translational regulation mediated by the ORFs, we examined translation following mutation of the initiation and termination codons of each of the three ORFs. We found that the ORFs marginally enhanced downstream gene expression. Moreover, repression of downstream gene translation was proportional to the lengths of the elongated ORFs and depended on the initiation contexts of the AUG codons. Although the ORFs play a major role in viral activities, their effects on translation were relatively minor. Rather, the ORFs may affect the fate of unspliced avian retroviral RNA in chronically infected cells by participating in the sorting of viral RNA for either translation or encapsidation into virions.

Animals↗
PubMed1993

Enhanced incorporation of transgenic DNA into zebrafish chromosomes by a retroviral integration protein.

Manufacture of lines of fish containing specific transgenes is difficult because most fish that hatch from embryos injected with foreign DNA are mosaic; few have the transgenic DNA integrated in germ-line cells. To determine whether the process of integration of exogenously supplied DNA into fish genomes could be accelerated, we examined the ability of the Moloney murine leukemia virus (MoMLV) integration protein (IN) to function in embryonic zebrafish cells. We used partially purified IN from a baculovirus/insect cell expression system and unpurified IN from extracts of psi-2 mouse cells that carry a MoMLV provirus. Both forms of IN were able to enhance expression in zebrafish 10 days after fertilization. At day 14 of development, fish injected with IN had higher levels of transgenic DNA than control fish. The ability of IN to enhance integration of transgenic constructs was demonstrated by a ligation-mediated polymerase chain reaction procedure, which was employed to detect junction fragments of foreign and host genomic DNA, generated by IN-mediated integration.

Animals↗
PubMed1992

Molecular cloning and sequence analysis of the cDNA for northern pike (Esox lucius) growth hormone.

The complete nucleotide sequence of the northern pike (Esox lucius) cDNA for pregrowth hormones was determined from clones derived from a pituitary gland cDNA library. Seventeen cDNA clones were isolated from a single mRNA species. A cDNA of 1,227 nucleotides was sequenced and found to encode a polypeptide of 209 amino acid residues, which included a putative signal sequence of 22 amino acid residues. Sequence comparison of the northern pike growth hormone gene to other known growth hormone genes revealed similarities closest to other members of the superorder Protacanthopterygii, which includes the Salmonidae family (i.e., salmon and trout).

Amino Acid Sequence↗
PubMed1992

Characterization of AluI repeats of zebrafish (Brachydanio rerio).

Two families of repetitive DNA sequences were isolated from the zebrafish genome and characterized. Eight different sequences were sequenced and classified by two standards, their (G + C) composition and their lengths. For convenience, the sequences were first divided into two types. Type I was (A + T)-rich, was repeated approximately 500,000 times, and constituted approximately 5% of the zebrafish genome. Type II was (G + C)-rich, was reiterated approximately 90,000 times, and comprised approximately 0.5% of the genome. Agarose gel electrophoresis of zebrafish DNA cleaved with AluI revealed three distinguishable bands of repetitive fragments: large (approximately 180 bp, designated RFAL), medium (approximately 140 bp, RFAM), and small (approximately 90 bp, RFAS). The RFAL fragments contained both type I and type II sequences. Limited digestion of genomic DNA indicated that RFAL and RFAM were tandemly arranged in the genome, whereas RFAS showed a mixed pattern of both tandem and interspersed repeated arrangements. Although inclusion of a repetitive sequence in a transgenic construct did not appreciably accelerate homologous integration of transgenes into the zebrafish genome, the AluI sequences could facilitate transgene mapping following chromosomal integration.

Animals↗
PubMed1991

Phylogenetic and physical analysis of the 5' leader RNA sequences of avian retroviruses.

A study of the secondary structures of the 5'-leader RNA sequences of avian leukosis/sarcoma viruses was conducted using phylogenetic sequence alignment, theoretical structures calculated from base-pairing interactions involving the calculated minimal delta G values, and RNaseT1 sensitivity. The results suggest that all of the avian retroviral RNA leaders may be able to adopt similar conformations. Open reading frames in the leader RNAs may be positioned to facilitate viral activities such as translation and packaging of the genomic RNA into virus particles.

Avian Leukosis Virus↗
PubMed1990

Development of expression vectors for transgenic fish.

Genetic alteration of fish is important for aquatic biotechnology as well as for investigating molecular interactions that occur during vertebrate development. The numerous, large, transparent, and externally fertilized eggs of many fish species make them ideally suitable for genetic manipulation, especially for production of transgenic animals. Genetic engineering of fish requires suitable expression vectors. Accordingly, we developed two fish expression vectors, FV-1 and FV-2, which contain the proximal promoter and enhancer regulatory elements of the carp beta-actin gene and the polyadenylation signal from the salmon growth hormone gene. The two fish expression vectors were tested in microinjected fish eggs and in tissue cultured fish and mammalian cells. These two "all-fish" expression vectors should be useful for genetic engineering of fish and have been used with growth-enhancing genes in transgenic fish.

Actins↗
PubMed1990

Functional analysis of elements affecting expression of the beta-actin gene of carp.

Regulatory regions of the beta-actin gene of the common carp (Cyprinus carpio) have been examined by linking upstream, 5'-flanking sequences and regions of the first intron to a bacterial chloramphenicol acetyltransferase (CAT) reporter gene. By analysis of the mRNA products and encoded CAT activity, we have identified four putative regions that influence expression: (i) a negative regulatory region 2,300 to 1,100 base pairs (bp) ahead of the gene; (ii) a proximal promoter element, containing the highly conserved CCAAT, CC(A/T)6GG, and TATA boxes, that is within the first 204 bp upstream of the initiation site; (iii) a negative element of 426 bp in the 5' region of the first intron; and (iv) a positive 304-bp element near the end of the first intron that contains highly conserved sequences found in all characterized beta-actin genes. The positive intron element is not a classical enhancer; it is position and orientation dependent, as has been observed in other housekeeping genes in vertebrates. Depending on the elements joined together, CAT gene expression can be modulated more than 500-fold in transfected mouse cells.

Actins↗
PubMed1990

Isolation and characterization of beta-actin gene of carp (Cyprinus carpio).

A beta-actin gene of carp (Cyprinus carpio) was isolated from a genomic EMBL3 library. The nucleotide sequence of the gene indicates six exons spanning 3.6 kb. Southern blot hybridization of restriction endonuclease digests of carp genomic DNA indicate that there are two copies of the beta-actin isotype and several other species of actin genes. The transcriptional start site is 85 bp and 24 bp downstream respectively from consensus CCAAT and TATA promoter elements. The organization of the carp beta-actin gene is identical to that of chicken, human, and rat genes in terms of size, exon/intron locations and junctions and in having a translationally silent first exon. The fish gene is 90% and 99% conserved at the nucleotide and amino acid levels, respectively, with land vertebrate beta-actin genes. Northern blot analysis of beta-actin gene expression indicated that the gene is highly expressed in brain, less so in muscle, and much less so in liver cells. The putative beta-actin proximal promoter of carp, identified by the conservation of known actin regulatory sequences, is transcriptionally active in both mammalian and piscine cells.

Actins↗
PubMed1989

A mutation in the short 5'-proximal open reading frame on Rous sarcoma virus RNA alters virus production.

The 5'-proximal open reading frame on Rous sarcoma virus RNA encodes a seven-amino-acid peptide and is conserved in all avian sarcoma-leukosis retroviruses. Ribosome-binding site analysis in intact chick cells showed that the 5'-proximal AUG codon is a strong site for initiation of translation in vivo. Removal of the 5'-proximal AUG codon by site-specific mutagenesis resulted in a virus with a reduced ability either to replicate or to transform a population of chicken embryo fibroblasts. These results establish a procedure for determining sites of ribosome binding and initiation of translation on mRNAs in intact eucaryotic cells and strongly suggest that the 5'-proximal open reading frame (or its AUG codon) on Rous sarcoma virus RNA has an important role in regulating viral activity.

Animals↗
PubMed1989

Effects of alterations in the leader sequence of Rous sarcoma virus RNA on initiation of translation.

The 372-nucleotide leader sequence of Rous sarcoma virus RNA contains three conserved short open reading frames and other sequences responsible for a variety of life cycle functions. We have investigated several aspects of the leader RNA which may influence the translation of the major coding regions to which the leader is juxtaposed. We found that small perturbations of the leader length do not affect the binding and scanning of ribosomal subunits by more than about 10%, that the length and/or structure of the RSV RNA leader is near optimal for translation of the major coding regions of the viral RNA, that inclusion or deletion of open reading frames influences downstream initiation in a manner that is not strictly additive, and that reinitiation of translation at the gag gene is very efficient.

Avian Sarcoma Viruses↗