A simplified method for in vivo footprinting using DMS.
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Alkylating Agents↗
Biomedical subjects
R K Patient
Publications and source records attributed to R K Patient.
Control of gene expression: tissue-specific expression.
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Animals↗
Transcriptional regulation of two cytotoxic T lymphocyte-specific serine protease genes.
The expression of two serine proteases is induced by antigenic stimulation in cytotoxic T lymphocytes. Using nuclear run-on analysis the increase in steady state mRNA level has been shown to correspond to transcriptional activation. However, the two genes appear to be sequentially rather than coordinately induced. Both genes were shown to be more sensitive to DNase I digestion than a beta-globin gene in cytotoxic T cells. In addition, for the cytotoxic cell protease 1 gene the 5' region of the gene was more sensitive than the 3' end. Two DNaseI hypersensitive sites were seen in the 5' flanking sequences of both genes. The DNA sequences of the upstream regions of both genes were determined and compared. Although the two flanking sequences are overall quite dissimilar, there are short regions which are shared between the two CTL-protease genes. A number of these have been implicated in regulating the expression of other T cell genes.
Animals↗
Structure and expression of a newt cardio-skeletal myosin gene. Implications for the C value paradox.
As part of our studies on the fate of the muscle lineage during amphibian limb regeneration, we have isolated genomic and cDNA sequences from a myosin heavy chain in the newt (Notophthalmus viridescens). Notwithstanding the technical problems inherent in analysing the large newt genome, genomic and cDNA sequences have been isolated and subjected to analysis by restriction mapping. Northern hybridization, Southern hybridization and DNA sequencing. We believe these to be the first single copy newt gene sequences to have been subjected to this type of analysis. The newt gene sequences showed a striking difference from mammalian myosins in both the estimated sizes of the gene and its intervening sequences; these being much larger than in the mammalian models, it is speculated that this could contribute to the exceptional size of the newt genome. By contrast, the coding sequences displayed very high levels of sequence homology to mammalian myosins. In particular, the amino acid sequence of the newt myosin was found to have greatest homology with rat and human myosin isotypes having a similar cardio-skeletal muscle expression pattern. Despite a long evolutionary separation, newt and mammalian cardio-skeletal myosins have remained more similar to each other than have the human or rat cardiac forms to skeletal myosins within their own respective species.
Amino Acid Sequence↗
5' structural motifs and Xenopus beta globin gene activation.
We have analysed the structure of the Xenopus beta globin gene 5' flanking region in erythroid and non-erythroid chromatin, in supercoiled plasmids and in minichromosomes assembled in HeLa cell transfections. We have identified two erythroid chromatin-specific, nuclease-hypersensitive sites (HSs), one centred on the cap site, the other located 1000 base-pairs further upstream. An (AT)n tract is located 200 base-pairs upstream from each of these sites. In supercoiled plasmids, the (AT)n tracts, and not the chromatin HSs, are preferentially cleaved by single strand and double strand-specific nucleases. Using restriction enzymes, we have looked at the structure of the cap site HS in minichromosomes assembled in HeLa cell transfections. We find that the structure is indistinguishable from that found in erythroid chromatin, thus reinforcing our previous suggestion, based only on DNase I studies, that the formation of this HS is not dependent on erythroid-specific factors. In view of this close structural mimicry of the situation in vivo, we have used the HeLa cell model system to study the sequences required for cap site HS formation. We find that deletion of the (AT)n tract immediately upstream influenced neither the formation of the HS nor transcription of the globin gene. Indeed, these features remained unaffected by further deletion of upstream sequences, including 50 base-pairs of the HS itself. In this construct, the dimensions of the HS remained the same as in the undeleted construct, with the plasmid sequences that replaced the deleted Xenopus sequences becoming hypersensitive. Thus, HS formation is directed by sequences downstream from --116 acting over a distance of at least 50 base-pairs.
Animals↗
Role for DNA replication in beta-globin gene activation.
Transcriptional activation of the Xenopus laevis beta-globin gene requires the synergistic action of the simian virus 40 enhancer and DNA replication in DEAE-dextran-mediated HeLa cell transfections. Replication does not act through covalent modification of the template, since its requirement was not obviated by the prior replication of the transfected DNA in eucaryotic cells. Transfection of DNA over a 100-fold range demonstrates that replication does not contribute to gene activation simply increasing template copy number. Furthermore, in cotransfections of replicating and nonreplicating constructs, only replicating templates were transcribed. Replication is not simply a requirement of chromatin assembly, since even unreplicated templates generated nucleosomal ladders. Stimulation of beta-globin transcription by DNA replication, though less marked, was also observed in calcium phosphate transfections. We interpret these results as revealing a dynamic role for replication in gene activation.
Animals↗
Evidence for dedifferentiation and metaplasia in amphibian limb regeneration from inheritance of DNA methylation.
Amphibian limb regeneration is a process in which it has been suggested that cells of one differentiated type may dedifferentiate and give rise to cells of another type in the regenerate. We have used two tissue-specific hypomethylations in the newt cardioskeletal myosin heavy chain gene as lineage markers to follow the fate of cells during limb regeneration. Analysis of genomic DNA from different muscle cell populations allowed the assignment of one marker to the muscle (Hypo A) lineage and the other, more tentatively, to the 'connective tissue' (Hypo B) component of muscle. The contribution to regenerated limb cartilage and limb blastemal tissue by cells carrying these markers was estimated by quantitative analysis of Southern blot hybridizations using DNA from regenerate tissues. The results are consistent with a contribution of cells from both muscle and connective tissue lineages to cartilage in regenerated limbs. In addition, removal of the humerus at the time of amputation (eliminating any contribution from pre-existing cartilage), has provided evidence for an increased representation of cells carrying the connective tissue marker in regenerate cartilage but did not affect the representation of cells carrying the muscle cell marker.
Animals↗
Highly efficient beta globin transcription in the absence of both a viral enhancer and erythroid factors.
We have studied the transcription of the Xenopus major adult beta globin gene in microinjected Xenopus oocytes at various levels of injected template, with or without the SV40 enhancer. We find that enhancer-independent transcription is highly efficient, being only two orders of magnitude below the calculated in vivo rate. Linkage to the SV40 enhancer has very little stimulatory effect. We have also tested the effect of replication on transcription in the oocyte system where replication was induced by progesterone treatment followed by prick activation. We found that the presence of replicated templates did not stimulate expression of the Xenopus beta globin gene either in the presence or absence of the SV40 enhancer. In addition, we found that specificity of transcription, in cryptic of initiation at the cap site versus initiation at cryptic promoters upstream of the cap site, was dramatically improved by the injection of higher numbers of beta globin templates, by oocyte maturation and activation or by the presence of the SV40 enhancer.
Animals↗
RecBC, sbcB independent, (AT)n-mediated deletion of sequences flanking a Xenopus laevis beta globin gene on propagation in E. coli.
Plasmids containing sequences 3' of the adult beta 1 globin gene of Xenopus laevis are unstable on propagation in a range of E. coli host strains. Up to 300 bp of Xenopus DNA are lost by rec A independent recombination between (AT)37 and (AT)17 sequences. Additionally, smaller deletions occurring in or around the (AT)37 sequence are observed. Deletion of these potential cruciform structures occurs in the absence of exonuclease I, exonuclease V and exonuclease VIII as the same pattern of deletion events is observed in recA recBC sbcB and recBC sbcA recE strains.
Animals↗
Facile cruciform formation by an (A-T)34 sequence from a Xenopus globin gene.
We have studied the structure adopted by an (A-T)34 sequence from a Xenopus globin gene when present in a negatively supercoiled plasmid. A variety of enzyme and chemical probing experiments and electrophoretic migration shift methods reveal that the sequence adopts cruciform geometry at moderate levels of supercoiling. The structure has the lowest free energy of formation yet observed for a cruciform, and no detectable kinetic barrier preventing rapid interconversion between extruded and unextruded conformations. Analysis of band-shift experiments reveals a twist change on cruciform formation of -5.8, slightly smaller than the -6.5 we would predict on the basis of a transition from B DNA. An attractive explanation consistent with this discrepancy is that the (A-T)34 stretch is locally underwound to about 11.7 base-pairs/helical turn at low levels of supercoiling. This calculation is made on the assumption that the cruciform junction is structurally similar to those examined previously, which is supported by the nuclease digestion results. This perturbed helical structure could be of considerable biological significance.
Acetaldehyde↗
(AT)n is an interspersed repeat in the Xenopus genome.
We have observed (AT)34 and (AT)23 tracts close to the coding sequences of the Xenopus laevis tadpole alpha T1 and adult beta 1 globin genes, respectively. We show that (AT)n sequences are found as interspersed repeats within the Xenopus globin and histone gene loci. Using (AT)n co-polymer in filter hybridisation experiments we estimate that there are 10(4) (AT)n tracts per haploid Xenopus genome. Hybridisation to genomic blots of DNA from yeast, slime mold, trypanosome, fruit fly, salmon, chicken, rat, human, crab and Xenopus species shows that strictly alternating AT of sufficient length to hybridise appears to be most abundant in Xenopus and crab genomes. We show that the specificity of the co-polymer probe for strictly alternating AT is, however, dependent on the length of the probe. Hybridisation experiments using (TG)n copolymer suggest that this highly conserved repeat is found as clustered repeats in the Xenopus genome in contrast to other eukaryotic genomes so far studied.
Animals↗
The complete nucleotide sequence of the major adult beta globin gene of Xenopus laevis.
We present the complete nucleotide sequence of the major beta globin gene of Xenopus laevis with 127 nucleotides of sequence upstream of the gene and 212 nucleotides downstream. The start point of transcription was determined by S1 mapping and primer extension and the site of polyadenylation by comparison with the mRNA sequence determined from a cDNA clone. Comparison of the X. laevis sequence with other genes transcribed by RNA polymerase II reveals conserved features in the 5' flanking, 5' noncoding, intron/exon boundary, and 3' noncoding regions.
Amino Acid Sequence↗
Complete nucleotide sequence of a cloned cDNA derived from the major adult alpha-globin mRNA of X. laevis.
The complete sequence of a cloned cDNA derived from the major adult alpha-globin mRNA of Xenopus laevis (the South African Clawed Toad) is presented. The sequence contains the complete coding and 3' non-coding regions of the mRNA and part of the 5' non-coding region. The amino acid sequence of the encoded alpha-globin polypeptide has been deduced and is compared to other alpha-globin polypeptides. We find that the sequence is equally diverged from a bullfrog tadpole alpha-globin polypeptide and human alpha-globin polypeptide suggesting that these three sequences may have diverged from a common ancestral sequence several hundred million years ago.
Amino Acid Sequence↗
Replication, methylation, and expression of X laevis globin genes injected into fertilized Xenopus eggs.
In the South African clawed toad, Xenopus laevis, the tadpole alpha-globin gene (alpha T1) and the major adult alpha- and beta-globin genes (alpha 1 and beta 1) are linked in the genome in the order alpha T1-alpha 1-beta 1. We show that cloned DNA fragments containing the Xenopus globin genes are replicated after injection into newly fertilized Xenopus eggs, reaching a maximal copy number at gastrulation. The DNA initially replicates as supercoils, but later it is found in a high-molecular-weight form, comigrating with the chromosomal DNA. In contrast to what has been found in mouse eggs, DNA does not become methylated when injected into Xenopus eggs. If, however, DNA is methylated in vitro before injection, methyl groups are retained through replication with a high efficiency. Both the adult alpha 1- and beta 1-globin genes are transcribed from their correct promoters in early embryonic stages, but the amount of transcription is relatively low and decreases in parallel with the decline in the amount of DNA retained in the embryo.
Animals↗
The organization of the tadpole and adult alpha globin genes of Xenopus laevis.
Adult erythrocytes of X. laevis contain six electrophoretically resolvable globin polypeptides while tadpole erythrocytes contain four polypeptides, none of which comigrates with an adult protein. We show that three of the adult proteins are alpha globin polypeptides (alpha 1, alpha 2, alpha 3) and three are beta globin polypeptides (beta 1, beta 2, beta 3). We find that a tadpole alpha globin gene (alpha T1) is linked to the major adult locus in the sequence 5'-alpha T1-alpha 1-beta 1-3' with 5.2 kb separating alpha T1 from alpha 1. Another tadpole alpha globin gene (alpha T2) is linked to the minor adult locus in the sequence 5'-alpha T2-alpha 2-beta 2-3' with 10.7 kb separating alpha T2 from alpha 2. These linkage relationships are consistent with the major and minor loci having arisen by tetraploidization but the different separation of larval and adult globin genes at the two loci indicates the occurrence of some additional chromosomal rearrangement. Two alternative models are presented.
Amino Acid Sequence↗
The nucleotide sequence of the major beta-globin mRNA from Xenopus laevis.
The nucleotide sequence of a cloned fragment containing an almost complete copy of the mRNA encoding the major adult beta-globin polypeptide in Xenopus laevis, the South African Clawed Toad, is presented. A procedure for strand separation by hybridization to complementary mRNA was used to determine some of the sequence and this technique is described. The complete amino acid sequence of the polypeptide has been deduced and comparison with other vertebrate beta-globins reveals several highly conserved, and therefore potentially important, regions of the protein. The sequence of beta-globin mRNA has been determined in several mammals, and in the chicken. Thus we have searched for conserved regions in the non-coding portions of these mRNA sequences, which encode the same protein, but which have been evolving separately for several hundred million years.
Amino Acid Sequence↗
Molecular cloning of cDNA sequences coding for the major alpha- and beta-globin polypeptides of adult Xenopus laevis.
This report describes the synthesis and cloning of almost complete DNA copies of the mRNAs encoding the major alpha-globin and major beta-globin of X. laevis. Double-stranded globin cDNA was inserted into the PstI site of the plasmid pBR322 and two cloned recombinants (designated pXG6C1 and pXG8D2) were selected. These were shown to contain almost complete copies of X. laevis globin mRNA. Restriction enzyme maps were determined for each cDNA sequence using the established method of partial digestion of end labelled DNA. However, this procedure was modified such that isolation of individual DNA fragments was no longer required. Each plasmid was shown, by both hybrid arrested translation and filter selection of complementary RNA, to contain a sequence coding for one or other of the two major globin polypeptides. Sufficient DNA sequence information has been determined from each cDNA clone to demonstrate that pXG8D2 contains a beta-globin sequence and pXG6C1 contains an alpha-globin sequence.
Animals↗