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E Whitelaw

Publications and source records attributed to E Whitelaw.

At least 37 records · Page 2Linked to original sources
PubMed1993

Analysis of the human alpha-globin gene cluster in transgenic mice.

A 350-bp segment of DNA associated with an erythroid-specific DNase I-hypersensitive site (HS-40), upstream of the alpha-globin gene cluster, has been identified as the major tissue-specific regulator of the alpha-globin genes. However, this element does not direct copy number-dependent or developmentally stable expression of the human genes in transgenic mice. To determine whether additional upstream hypersensitive sites could provide more complete regulation of alpha gene expression we have studied 17 lines of transgenic mice bearing various DNA fragments containing HSs -33, -10, -8, and -4, in addition to HS -40. Position-independent, high-level expression of the human zeta- and alpha-globin genes was consistently observed in embryonic erythroid cells. However, the additional HSs did not confer copy-number dependence, alter the level of expression, or prevent the variable down-regulation of expression in adults. These results suggest that the region upstream of the human alpha-globin genes is not equivalent to that upstream of the beta locus and that although the two clusters are coordinately expressed, there may be differences in their regulation.

Animals↗
PubMed1992

The developmental regulation of the human zeta-globin gene in transgenic mice employing beta-galactosidase as a reporter gene.

We have investigated the developmental and tissue specific expression of the human embryonic zeta-globin gene in transgenic mice. A construct containing 550 bp of zeta-globin 5' flanking region, fused to a beta-galactosidase (lacZ) reporter gene and linked to the locus control region (LCR)-like alpha positive regulatory element (alpha PRE) was employed for the production of transgenic mice. Firstly, we compared the number of live born transgenic mice containing this construct to the number of live born transgenic mice containing the entire zeta-globin gene linked to the alpha PRE or the beta LCR. Data showed that 12% of mice generated from eggs injected with zeta-promoter/lacZ/alpha PRE DNA were transgenic compared to only 2% of mice generated from eggs injected with the entire zeta-globin gene linked to the alpha PRE or the beta LCR. The reduced number of live born transgenic mice containing the latter constructs suggests that death of transgenic embryos, possibly due to thalassaemia, may be occurring. X-gal staining of whole embryos containing the lacZ gene revealed that zeta-globin promoter activity was most pronounced at 8.5-9.5 days of development and was restricted to erythroid cells. By 15 days of development, no zeta-globin promoter activity was detected. These results suggest that the alpha PRE can direct high level expression from the zeta-globin promoter and that sequences required for the correct tissue and developmental specific expression of the human zeta-globin gene are present within 550 bp's of 5' flanking region. Sequences within the body of the zeta-globin gene or 3' of the cap site do not appear to be necessary for correct zeta-globin developmental regulation.

Animals↗
PubMed1990

Regulated expression of globin chains and the erythroid transcription factor GATA-1 during erythropoiesis in the developing mouse.

Erythropoiesis in vertebrates is characterized by sequential changes in erythropoietic site, erythroblast morphology, and hemoglobin synthesis. We have examined the expression of globin chains and the major erythroid transcription factor GATA-1 (previously known as GF-1/NF-E1/Eryf 1) from days 7.5 to 17.5 of mouse development. mRNAs for embryonic (epsilon y2, beta H1, and zeta) and adult (alpha and beta) globin chains were quantitated by RNase protection assays. Switching of globins within the alpha-globin cluster (alpha and zeta) was not strictly coordinated with that within the beta-globin cluster (epsilon y2, beta H1, and beta). Regulation of globin switches during development was primarily transcriptional. Of particular note, we found two developmental switches (beta H1 to epsilon y2 and epsilon y2 to beta) in the mouse, more analogous than previously thought to shifts found in human development. The erythroid transcription factor GATA-1, believed to be a principal regulator of genes expressed in erythroid cells, first appeared in the embryo in yolk sac at the time of blood island formation and remained at a low level during embryonic erythropoiesis (8 to 11 days) relative to that found later in fetal liver (12 to 15 days). The rise in GATA-1 mRNA in fetal liver paralleled and preceded the rapid accumulation of adult beta-globin RNA. RNase protection assays and a GATA-1-specific peptide antiserum were used to establish that a single GATA-1 polypeptide is expressed throughout mouse development. Overall, these findings suggest that the levels of this erythroid transcription factor during development may contribute to the differential gene activation characteristic of definitive versus primitive erythropoiesis.

Animals↗
PubMed1989

Direct demonstration of termination signals for RNA polymerase II from the sea urchin H2A histone gene.

Previous studies [1,2] suggested but did not prove that the sea urchin H2A histone gene possesses strong transcriptional termination signals close to, but separate from, the 3' processing signals. In this study we have demonstrated by two independent approaches that these sequences elicit authentic transcriptional termination. First we show by nuclear run off analysis that nascent transcription terminates in the immediate 3' flanking region of the H2A gene, in an A-rich region. Second we show that these termination signals prevent transcriptional read through when placed in the intron of a globin gene. The intronic position of the termination signal rules out any effect on steady state mRNA levels. We have therefore defined DNA sequences which act as a transcription terminator when placed in heterologous RNA polymerase II genes.

Animals↗
PubMed1989

Transcriptional and translational analysis of the human theta globin gene.

The human theta-globin gene in man appears to be functional, based on its sequence and evolutionary conservation. However its physiological role is unknown and furthermore its deletion in some individuals appears to have no effect on erythroid development. We have therefore analysed the transcriptional and translational competence of the theta globin gene to assess whether or not it is a silent or active globin gene. First, we demonstrate that theta globin mRNA is correctly spliced, by sequencing its cDNA. Second, using this theta cDNA, we generated synthetic theta globin mRNA and were able to demonstrate that this mRNA is translated into theta globin protein in wheat germ in vitro translation extracts. Similarly, the theta globin gene transfected into an erythroid cell line produces a protein product that comigrates with theta globin. Finally, we analysed the unusual promoter of the theta globin gene. The GC rich sequence directly adjacent to the multiple cap sites of theta globin mRNA functions as a promoter element in both erythroid and non-erythroid cell lines, while the more usual CCAAT and ATA box regions (found in all other globin genes) which are displaced by the GC rich promoter sequence, do not possess detectible promoter activity. Taken together, these results suggest that theta globin may have some as yet undetermined role in human erythropoiesis.

Amino Acid Sequence↗
PubMed1989

Transcriptional promiscuity of the human alpha-globin gene.

The human alpha-globin gene displays the unusual property of transcriptional promiscuity: that is, it functions in the absence of an enhancer when transfected into nonerythroid cell lines. It is also unusual in that its promoter region lies in a hypomethylated HpaII tiny fragment (HTF) island containing multiple copies of the consensus sequence for the SP1-binding site. We have investigated whether there is a relationship between these two observations. First, we investigated the mouse alpha-globin gene since it does not lie in an HTF island. We have demonstrated that it was not transcriptionally promiscuous. Second, we studied the transcriptional activity of the human alpha-globin gene in the absence of the GC-rich region containing putative SP1-binding sites and found a small (two- to threefold) but consistent positive effect of this region on transcriptional activity in both nonerythroid and erythroid cell lines. However, this effect did not account for the promiscuous nature of the human alpha-globin gene. We found that in a nonreplicating system, the human alpha-globin gene, like that of the mouse, required a simian virus 40 enhancer in order to be transcriptionally active in nonerythroid and erythroid cell lines. Since we only observed enhancer independence of the human alpha-globin gene in a high-copy-number replicating system, we suggest that competition for trans-acting factors could explain these results. Finally, our experiments with the erythroid cell line Putko suggest that there are no tissue-specific enhancers within 1 kilobase 5' of the human alpha-globin cap site or within the gene itself.

Animals↗
PubMed1989

The globin switch at the level of mRNA in the developing mouse.

We have carried out a detailed analysis of the relative amounts of zeta, alpha, beta H1, epsilon y2 and adult beta globin mRNA in different tissues of the mouse embryo from Day 8.5 to Day 17.5 i.e. from the first signs of erythropoiesis until almost the end of gestation (birth is Day 19). Interestingly, we find that the zeta to alpha "switch" occurs 24 hours earlier in yolk sac than it does in fetal liver and that the ratio of zeta to alpha mRNA remains higher in the peripheral blood than in the yolk sac or the fetal liver during the latter half of gestation. In fact, zeta mRNA remains present in peripheral blood until Day 15.5. The switch at the mRNA level appears to mimic that found by others [Popp et al, 1987] at the protein level, at least for peripheral blood. This suggests that regulation is not occurring to any major extent at the translational level. The reiteration of the switch in fetal liver suggests that local environmental factors are involved. We find two switches within the beta cluster; beta H1 is expressed first, then epsilon y2 and lastly the two adult beta globins (beta major and beta minor). The switch from beta H1 to epsilon y2 occurs around Day 11.5 and the switch from epsilon y2 to the adult beta globins occurs around Day 15.5. This means that the pattern of expression of the beta-like globin genes in the mouse is more analogous to that found in the human than was previously thought.

Animals↗
PubMed1986

Globin gene transcripts can utilize histone gene 3' end processing signals.

Deletion of the poly(A) site from the human alpha globin gene results in a defective gene that produces very little stable mRNA as compared to the intact gene, presumably due to the instability of the mRNA. However, if the Alpha poly(A) site is replaced by mouse histone H4 3' end processing signals, significant levels of hybrid alpha/H4 mRNA are obtained and the transcripts formed are cytoplasmic and poly(A)-. When both mouse histone 3' end processing signals and the alpha globin poly(A) site signals are placed in tandem after the alpha globin gene promoter and coding sequence, the alpha poly(A) site signals are utilized exclusively. These results show that the histone 3' end processing signals can function independently of the histone promoter and the transcripts which are normally polyadenylated (alpha globin) can be stabilized by a poly(A)- histone mRNA 3' terminus. Furthermore, these results show that the histone 3' end processing signals are less efficient than the alpha globin poly(A) site signals, if the two are placed in direct competition.

Animals↗
PubMed1986

Alpha-thalassaemia caused by a poly(A) site mutation reveals that transcriptional termination is linked to 3' end processing in the human alpha 2 globin gene.

We have investigated the process of transcriptional termination in the duplicated human alpha globin genes which lie 4 kb apart on chromosome 16. In the human erythroleukemic cell line, K562, which expresses high levels of alpha globin, nuclear run-off experiments suggest that termination occurs within a region of 600 bp past the poly(A) site of both alpha 1 and alpha 2 globin genes. However, a thalassaemic alpha 2 globin gene with a non-functional poly(A) site AAUAAG, when transfected into HeLa cells, not only fails to 3' end process but also fails to terminate transcription. Studies on both steady-state RNA and nuclear run-off analysis of the primary transcripts show that transcription of the mutant alpha 2 globin gene reads through into the intergenic sequence past the normal termination site. These results demonstrate that transcriptional termination and 3' end processing of mRNA are coupled events for the alpha 2 globin gene.

Animals↗
PubMed1983

Transcriptional activity of the human pseudogene psi alpha globin compared with alpha globin, its functional gene counterpart.

Transcriptional analysis of the human pseudogene psi alpha globin has revealed the following features: (1) The promoter with a 23 bp deletion between the CCAAT and ATA boxes is functional both in vitro and in vivo, 3 fold and 10 fold less efficient, respectively, than alpha. (2) Both the psi alpha and alpha globin gene promoters are active in the absence of transcriptional enhancers, either a gene-encoded or viral enhancer. (3) The mutated poly(A) addition signal in psi alpha (AATGAA) appears to be completely nonfunctional. This result provides an explanation for the absence of psi alpha transcripts in human erythroid cells.

Base Sequence↗
PubMed1980

The detection and use of hemoglobin mutants in the direct analysis of human globin genes.

Many human globin-chain mutants contain amino acid replacements that result from single base changes in the corresponding globin gene. Using recombinants, the coding sequences of each of the alpha-, beta-, Ggamma-, and Agamma-globin genes have now been determined. Those sequences of DNA that are cleaved by a number of specific restriction endonucleases have been identified and accurately positioned. Mutations at these sequences abolish the restriction site, and therefore, the pattern of DNA fragments containing hybridizing globin-gene sequences is altered compared to DNA from normal persons. This allows the identification of one of a pair of cross-hybridizing human globin-gene sequences, as is shown here for the two alpha-globin, the two gamma-globin, and the delta- and beta-globin genes.

Amino Acid Sequence↗
PubMed1980

Mapping the alpha-globin genes in an Algerian HbH patient and his family.

The organization of alpha-globin genes in normal white European, normal Algerian, and alpha-thalassemic Algerian DNA was examined by restriction endonuclease mapping using HindIII, HpaI, Bamhi, EcoRI, BgIII, and PstI. The results for normal DNA confirm and add to the findings of Surrey et al. and Orkin; the two alpha-genes are approximately 3.0 kb apart. The restriction enzymes BgIII and HpaL cut between the two alpha-genes. Four PstL sites are located: two surrounding each alpha-gene. The physical maps for a number of Algerian controls (normal alpha- and beta-globin biosynthesis profiles) are identical to that of the European controls. The Algerian alpha-thalassemic presenting with HbH disease was found to be homozygous for a 3.5-3.7 kb deletion at the alpha-gene locus, leaving one alpha-gene per chromosome. The patient's mother and father are both found to be heterozygous for this deletion. An unaffected sibling carries both normal chromosomes. The deletion could be the result of a Lepore-like crossover fusion event between the two alpha-globin genes, or of a 3.7 kb deletion of either the entire 5' alpha-gene or the entire 3' alpha-gene. The Algerian case of HbH disease studied differs from Asian cases in both the mode of inheritance and the molecular pathology of the alpha-thalassemia mutation. If this type of deletion is the major cause of Algerian alpha-thalassemia, it would explain the apparent absence of Hydrops fetalis in this geographical area.

Algeria↗
PubMed1979

Regulation of ketogenesis during the suckling-weanling transition in the rat. Studies with isolated hepatocytes.

The rates of ketogenesis from endogenous substrates, butyrate or oleate, have been measured in isolated hepatocytes from suckling and weanling rats. Ketogenesis from endogenous substrate and from oleate decreased on weaning, whereas the rate from butyrate remained unchanged. It is concluded that the major site of regulation of ketogenesis during this period of development involves the disposal of long-chain fatty acyl-CoA between the esterification and beta-oxidation pathways. Modulators of lipogenesis [dihydroxyacetone and 5-(tetradecyloxy)-2-furoic acid] did not alter the rate of ketogenesis in hepatocytes from suckling rats, and it is suggested that this is due to the low rate of lipogenesis in these cells. Hepatocytes from fed weanling rats have a high rate of lipogenesis and evidence is presented for a reciprocal relationship between ketogenesis and lipogenesis, and ketogenesis, and esterification in these cells. Dibutyryl cyclic AMP stimulated ketogenesis from oleate in hepatocytes from fed weanling rats, even in the presence of an inhibitor of lipogenesis [5-(tetradecyloxy)-2-furoic acid], but not in cells from suckling rats. It is suggested that cyclic AMP may act via inhibition of esterification and that in hepatocytes from suckling rats ketogenesis is already maximally stimulated by the high basal concentrations of cyclic AMP [Beaudry, Chiasson & Exton (1977) Am. J. Physiol. 233, E175--E180].

Animals↗
PubMed1978

Physiological aspects of the regulation of ketogenesis.

The importance of ketone bodies (acetoacetate and 3-hydroxybutyrate) as substrates for peripheral tissues, especially nervous tissue, of man is now firmly established. This has renewed interest in the factors that control the production of ketone bodies by the liver in various physiological situations, such as alterations of dietary status, stage of development or alteration in demand for circulating substrates (e.g. in exercise or lactation). In the discussion of the regulation of ketogenesis in the present paper, distinction is made between extrahepatic and intrahepatic control. The former is mainly concerned with the factors (e.g. hormonal status of animals) that alter the flux of non-esterified fatty acids to the liver, whereas intrahepatic regulation involves the fate (esterification versus beta-oxidation) of fatty acids within the liver. Emphasis is placed on the fact that alterations in blood glucose concentrations are indirectly responsible, via effects on insulin secretion, for the extrahepatic control of ketogenesis. By analogy, it is postulated that the carbohydrate status of the liver may play a role in the intrahepatic regulation of ketogenesis. Some support for this postulate is provided by comparison of measurements of blood ketone-body concentrations in various inborn errors of hepatic carbohydrate metabolism (e.g. deficiencies of glucose 6-phosphatase, fructose 1,6-bisphosphatase and glycogen synthase) in man and by experiments with isolated rat hepatocytes. Present information on the short- and long-term factors that may be responsible for the altered rates of ketogenesis during the foetal-neonatal and suckling-weanling transitions, in lactation, on feeding a high-fat diet and post-exercise is discussed. It is concluded that the major factors involved in the regulation of ketogenesis in these situations are (a) flux of non-esterified fatty acids to the liver and (b) the partitioning of long-chain acyl-CoA between the esterification and beta-oxidation pathways.

Animals↗
PubMed1977

Effects of lactation of ketogenesis from oleate or butyrate in rat hepatocytes.

1. Rates of ketogenesis from endogenous butyrate or oleate were measured in isolated hepatocytes prepared from fed rats during different reproductive states [virgin, pregnant, early-lactating (2-4 days) and peak-lactating (10-17 days)]. In the peak-lactation group there was a decrease (25%) in the rate of ketogenesis from butyrate, but there were no differences in the rates between the other groups. Wth oleate, the rate of ketogenesis was increased in the pregnant and in the early-lactation groups compared with the virgin group, whereas the rate was 50% lower in the peak-lactation group. 2. Experiments with [1-(14)C]oleate indicated that these differences in rates of ketogenesis were not due to alterations in the rate of oleate utilization, but to changes in the amount of oleoyl-CoA converted into ketone bodies. 3. Although the addition of carnitine increased the rates of ketogenesis from oleate in all groups of rats, it did not abolish the differences between the groups. 4. Measurements of the accumulation of glucose and lactate showed that hepatocytes from rats at peak lactation had a higher rate of glycolytic flux than did hepatocytes from the other groups. After starvation, the rate of ketogenesis from oleate was still lower in the peak-lactation group compared with the control group. This suggests that the alteration in ketogenic capacity in the former group is not merely due to a higher glycolytic flux. 5. It is concluded that livers from rats at peak lactation have a lower capacity to produce ketone bodies from long-chain fatty acids which is due to an alteration in the partitioning of long-chain acyl-CoA esters between the pathways of triacylglycerol synthesis and beta-oxidation. The physiological relevance of this finding is discussed.

Animals↗