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J Mellor

Publications and source records attributed to J Mellor.

At least 55 records · Page 3Linked to original sources
PubMed1990

Chromosome rearrangements induced by recombinant coliphage lambda placMu.

Operon fusions to lacZ, commonly used to study bacterial gene expression in vivo, are normally constructed using phage derivatives such as lambda placMu53 or Mud-1. These derivatives contain a part of trp operon, and we have found that, when integrated into the chromosome, recombination can occur at high frequency between this trp DNA and the chromosomal trp operon leading to chromosomal inversions which fuse lacZ to the trp promoter. Large segments of the chromosome can be inverted by such rearrangements and their occurrence can seriously complicate the isolation of regulatory mutations and other studies unless appropriate precautions are taken. This phenomenon provides a simple means of isolating inversions of defined chromosomal segments and determining the direction of transcription of some lacZ operon fusions.

Bacteriophage lambda↗
PubMed1990

Characterisation of sequences required for RNA initiation from the PGK promoter of Saccharomyces cerevisiae.

In the phosphoglycerate kinase (PGK) gene of yeast, as in other highly expressed yeast genes, the sequences surrounding the site of RNA initiation have a loosely conserved structure of a CT rich stretch followed by the tetranucleotide CAAG. Using internal deletions and insertions we have identified the elements in the PGK promoter which are required for correct RNA initiation at the CAAG sequence at -39. The results indicate that two different components of the PGK promoter contribute to correct RNA initiation, the TATA homologies, located at -152 and -113, and the sequences at the site of initiation. Both TATA elements can function in RNA initiation. Deletion of the upstream TATA element, TATAI, results in slightly heterogeneous RNA initiation, but the majority of the RNA initiates correctly. Deletion of both the PGK TATA elements results in the majority of the RNA initiating at sites downstream from the wild-type I site, within the structural gene between +40 to +80. The CT rich box is not essential for correct mRNA initiation as shown by deletion analysis. The site of RNA initiation in the PGK promoter appears to be determined by sequences located immediately 5' of the CAAG sequence motif. This short sequence, ACAGATC, when located the correct distance from the TATA elements may be sufficient to determine a discrete initiation site.

Base Sequence↗
PubMed1990

CPF1, a yeast protein which functions in centromeres and promoters.

Centromeres and several promoters of Saccharomyces cerevisiae contain a highly conserved octanucleotide, RTCACRTG, called CDEI. Using biochemical, genetic and structural analyses, we show that the same protein binds in vivo to CDEI sites in centromeres and in promoters. This protein, called CPF1 for centromere promoter factor, binds DNA as a dimer. Inactivation of the gene is not lethal but leads to a partial loss of the centromere function and to a Met- phenotype. Changes of the chromatin structure due to inactivation of CPF1 are seen at centromeres and at several CDEI-carrying promoters (e.g. MET25, TRP1, GAL2). However promoter activities are affected in diverse ways making it presently difficult to describe a function for CPF1 in gene expression. The sequence of the cloned gene reveals in the carboxy-terminal part two potential amphipathic helices preceded by a positively charged stretch of amino acids very similar to the helix-loop-helix domains recently identified in factors controlling tissue specific transcription in higher eukaryotes. Carboxy-terminal truncations of CPF1 lacking this domain no longer bind to CDEI. The amino-terminal half of CPF1 carries two clusters of negatively charged amino acid residues. Surprisingly, deletions of these clusters still render cells Met+ and lead only to a marginal decrease in centromere activity.

Amino Acid Sequence↗
PubMed1988

An AT rich region of dyad symmetry is a promoter element in the yeast TRP1 gene.

Transcription from the yeast TRP1 promoter results in two classes of transcript, I and II, that are influenced by different promoter elements. The 5' flanking region contains a region of dyad symmetry (RDS) which contains a 12 nucleotide AT rich inverted repeat, separated by a 21 bp spacer region. The RDS lies within a region of the promoter required for transcription of class II RNAs. A series of internal deletions and insertions have been constructed in vitro around the RDS and the effect of each mutation on transcription has been analysed. Deleting either of the repeats abolishes class II transcription and disruption of both repeats influenced the levels of the larger class I transcripts. Deletion of the spacer had no effect but increasing the length to 33 bp reduced transcription. These results show that the RDS is an important component of the TRP1 promoter, that both repeats must be preserved and that there is some constraint on the spacing of the repeats for maximal function.

Base Sequence↗
PubMed1988

Pulmonary function testing in predicting complications from percutaneous lung biopsy.

Percutaneous needle biopsy is an accepted method of obtaining tissue for diagnosis of lung tumors. The depth of the lesion, size of the needle, operator experience, and the presence of emphysema have been identified as factors influencing the risk of postbiopsy pneumothorax, the most common complication. In this retrospective study of 308 patients, we enquired whether pulmonary function tests (available in 138 patients) and arterial PO2 (available in 103 patients) might predict the risk of pneumothorax following percutaneous needle biopsy. We found that as airway obstruction increases (FEV1.0/FVC less than 59% of predicted) or as arterial oxygenation decreases (PO2 less than 59 mm Hg), not only does the incidence of pneumothorax increase, but symptoms are more severe in that the number of pneumothoraces requiring chest tube drainage increases as well. We suggest that airway obstruction and arterial oxygenation are factors indicative of increased risk identifying patients who need close scrutiny after the procedure.

Airway Obstruction↗
PubMed1987

The UAS of the yeast PGK gene contains functionally distinct domains.

The upstream activation site (UAS) of the yeast phosphoglycerate kinase gene (PGK) has been localised by deletion analysis (1). Here we show that the UASPGK contains two functionally distinct domains. These two domains, designated activator (A) and modulator (M), appear to be located within bases -460 to -402 and -531 to -461, respectively, relative to the initiating ATG; although it is possible that part of the M domain resides within the A domain. They have been shown, using a heterologous assay promoter, to have distinct transcriptional functions. Domain A is responsible for activation of transcription whilst domain M is required for carbon source dependent regulation of transcription. Protein-DNA binding studies have demonstrated that the DNA fragment containing domain M has high affinity for at least one specific DNA-binding protein, whilst domain A does not appear to interact strongly in protein-binding assays under the same conditions. The domain M binding activity is dependent on the carbon source in the growth medium and may be functional in the carbon source control of PGK expression.

Base Sequence↗
PubMed1987

A transcriptional activator is located in the coding region of the yeast PGK gene.

Expression of heterologous genes from the PGK promoter on high copy number plasmids in yeast is relatively poor compared to the intact PGK gene because of low steady-state RNA levels. In this paper we show that low levels of heterologous RNA are not due to instability of mRNA but result from inefficient transcription due to a defect in RNA synthesis. A comparison of RNA levels from homologous and heterologous transcription units allowed the identification of a positive activator for transcription within the PGK coding region which is required for efficient expression of the PGK gene. Deletion of this region, the "downstream activator sequence", causes a six to ten fold reduction in transcriptional efficiency from the PGK 5' noncoding region.

Base Sequence↗
PubMed1987

The functions and relationships of Ty-VLP proteins in yeast reflect those of mammalian retroviral proteins.

We have identified the major structural core proteins of Ty virus-like particles (Ty-VLPs) and shown that they are generated by proteolytic cleavage of the primary translation product of TYA, p1. This precursor protein is therefore functionally similar to the gag precursor of retroviruses. Cleavage is mediated by a Ty-encoded protease located at the 5' region of TYB and is accompanied by a change in particle morphology. p1 contains sufficient information for the assembly of a pre-Ty-VLP complex, which does not require the presence of either Ty protease or reverse transcriptase. The results indicate that the requirements and pathway of Ty-VLP formation reflect the initial stages of mammalian retroviral assembly and further support the idea of a common origin for Ty elements and retroviruses.

Amino Acid Sequence↗
PubMed1987

Adaptation of skeletal muscle to increased contractile activity. Expression nuclear genes encoding mitochondrial proteins.

An increase in mitochondrial biogenesis in mammalian cells requires a coordinated increase in the expression of a number of nuclear genes that encode mitochondrial proteins. To examine the regulatory mechanisms involved, we used specific anti-sense RNA probes to estimate the cellular concentrations of mRNA transcripts of two such nuclear genes in rabbit tibialis anterior muscles subjected in vivo to 10-21 days of indirect electrical stimulation. The unstimulated contralateral muscle in the same animals provided a base line for comparison. Change in expression of mitochondrial proteins was assessed in terms of the enzymatic capacity of citrate synthase and cytochrome oxidase, which increased 2.1-fold after 10 days and 5.5- and 4.1-fold, respectively, after 21 days of stimulation. As a proportion of total cellular RNA, messenger RNA encoding subunit beta of F1-ATPase increased 2.2-fold over control levels after 10 days and 2.3-fold after 21 days; mRNA encoding subunit VIC of cytochrome oxidase increased 1.3-fold and 1.9-fold over control levels after stimulation for 10 and 21 days, respectively. These changes were not attributable to nonspecific effects of stimulation on all mRNA transcripts, since aldolase A mRNA decreased to 26% of control levels after 21 days of stimulation. Furthermore, mRNA transcripts from these nuclear genes encoding mitochondrial proteins did not increase to the same extent as mRNA transcripts of mitochondrial genes such as cytochrome b, which increased 5.9-fold after 21 days of stimulation. We conclude that the increase in mitochondrial biogenesis induced by electrical stimulation of skeletal muscle is supported by pretranslational regulation of expression of nuclear genes encoding mitochondrial proteins. There are, however, indications that translational or post-translational regulatory events may also be involved.

Adaptation, Physiological↗
PubMed1987

The genetic organization of the yeast Ty element.

The genetic organization of the yeast transposon Ty resembles that of higher eukaryotic retroviruses and other elements such as the copia-like sequences of Drosophila. The Ty genome is 5.9 kb (10(3) bases) long. It has 340 bp (base pairs) terminal repeats known as delta sequences and it produces a terminally redundant 5.7 kb RNA that starts in the 5' delta and ends in the 3' delta. Ty transcription is directed by signals upstream and downstream of the major RNA start site and is regulated by the mating-type configuration of the cell. The 5.7 kb transcriptional unit is divided into two overlapping open reading frames, TYA and TYB. TYA occupies approximately the first quarter of the transcriptional unit while TYB occupies the rest. TYB overlaps TYA by either 38 or 44 nucleotides, depending on the element, and is in the plus one reading frame with respect to TYA. TYA is expressed to produce protein p1 (50 x 10(3) Mr) and TYB is expressed as a TYA:TYB fusion protein, p3 (190 x 10(3) Mr). Both of these proteins are subsequently cleaved to produce proteins p2, p4, p5, p6, reverse transcriptase and a protease that is responsible for some of these cleavage events. These proteins are assembled into virus-like particles (Ty-VLPs) that contain Ty RNA and reverse transcriptase activity. It is likely that the Ty-VLPs are units of transposition as Ty transposes via an RNA intermediate.

DNA Transposable Elements↗
PubMed1987

The organization and expression of the yeast retrotransposon, Ty.

The genome of the yeast Saccharomyces cerevisiae contains several copies of a mobile genetic element, Ty, which is representative of a group of eukaryotic transposable elements, retrotransposons. The organization and expression of Ty have several features in common with retroviral proviruses but they also show some important differences.

DNA Transposable Elements↗
PubMed1986

Expression strategies of the yeast retrotransposon Ty: a short sequence directs ribosomal frameshifting.

The Ty element of yeast is a member of a class of eukaryotic transposons which bear a striking resemblance to retroviral proviruses in their structure and expression strategies (1,2). A direct comparison can be drawn between the production of a fusion protein encoded by Ty, resulting from a frameshift event which fuses two out-of-phase open reading frames TYA and TYB, and the production of Pr180gag-pol in a retrovirus such as Rous Sarcoma Virus (RSV) (3,4). We present data which shows, definitively, that RNA splicing is not responsible for the frameshift in Ty. By in vitro mutation of a class I element, Ty1-15, we demonstrate that 31 nucleotides contained within the region where the TYA and TYB open reading frames overlap direct the frameshift. Within this short sequence there is a region of homology with a class II element which we show is also able to frameshift.

Base Sequence↗
PubMed1986

Regulation of nuclear and mitochondrial gene expression by contractile activity in skeletal muscle.

Increased contractile activity of skeletal muscle augments the volume fraction and enzymatic capacity of mitochondria and suppresses the enzymatic capacity of several cytoplasmic enzymes of glycolysis. To examine the biochemical mechanisms underlying these effects, we measured the concentrations of cytochrome b mRNA and aldolase A mRNA in tibialis anterior muscles of adult rabbits that had been stimulated via the motor nerve to contract continuously at 10 Hz for 5 or 21 days; these were compared with the corresponding levels in the unstimulated limbs of the same animals. After 21 days of stimulation aldolase mRNA had fallen to one-fourth of control levels, while cytochrome b mRNA had increased by 5-fold. A reduction in aldolase mRNA was already evident after only 5 days of stimulation, whereas the level of cytochrome b mRNA was not elevated at this stage. Mitochondrial DNA was unchanged after 5 days but had increased by 4-fold after 21 days. We conclude that contractile activity in skeletal muscle produces reciprocal changes in the expression of these two genes at a transcriptional level but via different regulatory mechanisms. Enhancement of the expression of the mitochondrial cytochrome b gene appears to be proportional to the increase in its copy number and may not, therefore, depend upon changes in transcriptional or translational efficiency. The reduction in aldolase A mRNA occurs at an earlier stage in the response to contractile activity and is probably mediated by a reduced transcriptional efficiency.

Animals↗
PubMed1986

Multiple control elements in the TRP1 promoter of Saccharomyces cerevisiae.

The TRP1 promoter generates two groups of mRNAs, transcript I and transcript II. The difference in size between the largest and smallest mRNAs is about 200 base pairs. A series of one-sided and internal deletions were constructed in vitro throughout the TRP1 promoter, and the effect of each deletion on transcription was assessed by Northern blotting. We showed that 395 base pairs of the TRP1 promoter were sufficient for the normal transcription of all RNAs and that the promoter contained two control domains. The control domain for transcript I consisted of one positive element and one negative element, while the control domain for transcript II contained two positive elements. The negative element, mapped between -293 and -318, expression of transcript I. Two regions of transcript I. Two regions (-280 to -236 and -235 to -209) were required for accurate initiation of transcript I. Each region contained sequences homologous to known consensus sequences of the TATA box.

Aldose-Ketose Isomerases↗
PubMed1986

Efficient expression of the Saccharomyces cerevisiae PGK gene depends on an upstream activation sequence but does not require TATA sequences.

The Saccharomyces cerevisiae PGK (phosphoglycerate kinase) gene encodes one of the most abundant mRNA and protein species in the cell. To identify the promoter sequences required for the efficient expression of PGK, we undertook a detailed internal deletion analysis of the 5' noncoding region of the gene. Our analysis revealed that PGK has an upstream activation sequence (UASPGK) located between 402 and 479 nucleotides upstream from the initiating ATG sequence which is required for full transcriptional activity. Deletion of this sequence caused a marked reduction in the levels of PGK transcription. We showed that PGK has no requirement for TATA sequences; deletion of one or both potential TATA sequences had no effect on either the levels of PGK expression or the accuracy of transcription initiation. We also showed that the UASPGK functions as efficiently when in the inverted orientation and that it can enhance transcription when placed upstream of a TRP1-IFN fusion gene comprising the promoter of TRP1 fused to the coding region of human interferon alpha-2.

Base Sequence↗