Biomedical subjects
Publications and source records attributed to J Davison.
An improved restriction site bank vector has been constructed from plasmid pJRD158. The new version is smaller and contains 43 unique restriction sites. It should greatly facilitate cloning versatility by providing unique sites for most commercially available restriction enzymes.
A comparison of interview data on 595 patients with newly incident cutaneous melanoma, excluding lentigo maligna melanoma and acral lentiginous melanoma, with data from comparison subjects drawn from the general population, showed that melanoma risk increased in association with the frequency and severity of past episodes of sunburn, and also that melanoma risk was higher in subjects who usually had a relatively mild degree of suntan compared to those with moderate or deep suntan in both winter and summer. The associations with sunburn and with suntan were independent. Melanoma risk is also increased in association with a tendency to burn easily and tan poorly and with pigmentation characteristics of light hair and skin colour, and history freckles; the associations with sunburn and suntan are no longer significant when these other factors are taken into account. This shows that pigmentation characteristics, and the usual skin reaction to sun, are more closely associated with melanoma risk than are sunburn and suntan histories.
The related phenomena of ColE1 DNA replication control and plasmid incompatibility are analysed in detail. Replication is negatively regulated in different ways by two gene products, RNA I and gene rop-coded polypeptide, while plasmid incompatibility is specified only by RNA I. Thus, copy-number mutants in the RNA I region usually have altered incompatibility and vice versa. RNA I is transcribed from the opposite strand to RNA II; the processed RNA II transcript serves as the primer for DNA replication. The base-pairing interaction between RNA I and RNA II controls DNA synthesis and incompatibility by preventing primer formation. A model is proposed to explain the phenotype of RNA I mutants in terms of the changes they cause in the secondary structure of RNA I or RNA II.
A DNA fragment has been constructed that contains many unique cloning sites not present in currently used Escherichia coli plasmid cloning vehicles. Insertion of this fragment into a modified version of pBR322 results in an AmpRTetR vector (pJRD158) of 3903 bp containing 28 unique cloning sites, four "almost unique" cloning sites, and eight unassigned unique 6-bp palindromes. The plasmid has the additional advantages of very high copy number and altered incompatibility. The latter permits it to be stably maintained in the same host as pBR322.
pJRD158 is a small plasmid vector (3903 bp) derived from pBR327 and specifying resistance to ampicillin and tetracycline. It contains 28 unique restriction sites (and 4 nonunique restriction sites) that can be used for cloning. The DNA sequence and computer-assisted restriction site analysis of pJRD158 are reported. Evidence is also presented that suggests a 2-bp revision of the DNA sequence of pBR322 in the RNA primer region.
Plasmids are described that allow fusions between the Escherichia coli galK gene (coding for galactokinase) and any gene of interest. An example is given in which a galK gene, lacking the normal initiator methionine codon, is fused to various segments of the 5' end of the tetR gene of pBR322. The resulting plasmids complemented an E. coli galK mutant, and galactokinase activity was retained despite the addition of up to 250 foreign amino acids to the amino-terminus of the galactokinase polypeptide. In a second experiment, the galK gene was fused to the LEU2 gene of Saccharomyces cerevisiae. The resulting plasmid was able to complement a yeast GAL1-mutant and galactokinase synthesis in yeast was controlled, via the LEU2 regulatory system, by the levels of leucine and threonine in the growth medium. The galK fusion plasmids should facilitate analysis of the control systems of a wide variety of genes in different organisms.
The transcription of the T5 first-step transfer genes A2 and A3 has been shown to proceed from right to left (i.e., from A3 to A2) on the T5 DNA molecule.
Transcription promoters and terminators have been cloned from the late region of bacteriophage T5 DNA and their strengths determined in vivo in plasmid derivatives. DNA sequence analysis shows these transcription signals to be remarkable in that, in all four cases studied in detail, the promoters and terminators overlapped or were very close together.
Cloning into the HindIII site of plasmid pBR322 inactivates the tetR promoter and usually prevents the expression of the tetR gene. The corresponding clones revert to tetracycline resistance at a low frequency. Such reversion is caused by gene rearrangement within the plasmids. DNA sequence analysis reveals three classes of revertants. The first class contains plasmids with partial duplications, which result in the fusion of the promoter of the RNA I species to the tetR gene. The event itself destroys the region encoding the RNA primer for replication and thus the plasmids would be replication defective if the duplication did not also include this region of the molecule. The plasmids from the second class are simple deletions which again fuse the tetR region to the RNA I promoter. In one case, the junction takes place at the end of the RNA I transcript, leaving RNA I and the RNA primer virtually intact. However, it removes the promoter of the RNA primer, the latter now being read from the cloned material. The second member of this class has fused the tetR gene well upstream of the RNA I region so that the RNA primer is still read from its own promoter. The low-level tetracycline resistance is probably due to partial read-through of the RNA I terminator. The third class of revertants differs from the previous two by the acquisition of foreign DNA in the form of an IS2-type insertion element which is known to promote transcription.
A general method is described that allows precise deletion of a chosen restriction fragment(s) from a plasmid having many cleavage sites for that restriction enzyme. The DNA to be deleted is first separated from the rest of the plasmid on a larger DNA fragment contained between two different unique restriction sites. This fragment is then subdigested by the restriction endonuclease of interest, which recognises two or more tetranucleotide (cohesive end or blunt end) sequences on the fragment, and is recloned between the two original unique restriction sites. The method is rapid, efficient, and the results are predictable. Examples are given in which predetermined HpaII (9 bp, 147 bp), TaqI (141 bp) and AluI (15 bp, 403 bp) fragments have been selectively removed from the tetR region of plasmid pBR322.
An experiment is reported that explored the role of self-attributional differences as mediators of reactions to threats to control in Type A and Type B individuals. Both Type A and Type B subjects were placed in a choice elimination reactance paradigm. In addition to the standard control and choice elimination conditions, subjects were induced to attribute having a choice among evaluated items either to themselves or to the situation prior to experiencing the elimination of choice. Reactance was measured by the subjects' reevaluations of the eliminated choice alternative. Results indicated that when a behavioral freedom is blocked, without manipulating attributions, only Type As experienced reactance. When subjects were induced to attribute having a choice to themselves, the magnitude of the reactance responses increased for both Type As and Type Bs. In contrast, when Type As and Type Bs were induced to attribute having choice to the situation, neither group reacted to the loss of choice. These results are integrated with previous findings on the coronary-prone behavior pattern.
It has been proposed (Hoeijmakers et al., 1980b) that variant surface antigen (VSA) gene expression in Trypanosoma brucei is accomplished by a gene re-arrangement involving the basic copy of the VSA gene to give the so-called expression-linked copy (which is present only in the strain expressing that particular antigen). In this publication, the basic and expression-linked copies of the gene have been visualized by Southern blot analysis of nuclear DNA and shown to be located on HindIII fragments of 4.5 and 10-12 kb, respectively. In addition, several other bands of weaker hybridization are seen, probably representing evolutionary relatives. Using a shotgun approach, HindIII gene banks have been constructed and recombinants isolated which carry the 4.5-kb HindIII fragment containing the VSA118 gene basic copy. Several clones containing evolutionary relatives were also found. The 4.5-kb HindIII fragment is able to hybridize to probes derived from both the 5' and 3' ends of the cDNA, while the relatives have homology only to the 3' end. A detailed comparison of the restriction map of VSA118 cDNA with that of the VSA118 basic copy showed no differences, demonstrating that the gene contains no introns. This result also indicates that the gene from which VSA118 mRNA is transcribed (whether this be the basic copy or the expression-linked copy) is identical to the basic copy over the region analysed.
A new multipurpose transthoracic pacing device is described that will simultaneously enable transthoracic electrode insertion, intracardiac drug infection, and blood sample removal for blood gas determination, all via a single myocardial needle insertion. The device proved efficacious and safe in trials in an animal model. In clinical use in 10 emergency room patients with brady-asystolic cardiac arrest, pacing was achieved in all 10 with one short-term survivor. Median insertion time was 30 seconds. Median threshold in 6 patients was 2.5 mA (1 to 6). This new device enables reliable and rapid emergency treatment of brady-asystolic cardiac arrest.
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The adjacent PstI-J, I and G fragments of the phage T5 DNA molecule (4.4, 4.6 and 7.2 kb, respectively) have been cloned in plasmid pBR322 and their locations verified by Southern blot analysis. The PstI I and G fragments overlap the previously cloned HindIII-P and G fragments and like those, contain no known genetic markers. In addition, one of the 12 newly isolated T5 mutants maps in this PstI-IG region. Thus, the size of the "empty" region between genes D15 and D17, which we have previously observed on the genetic map, extends to at least 11.8 kb. In contrast, the PstI-J fragment carried part of the D12 gene and the intact D14 and D15 genes. This clone is of particular interest since the D15 gene product is a nuclease and is responsible for the positive control of late gene transcription. The orientation of these genes relative to the T5 DNA molecule has been determined by a combination of restriction, deletion and complementation analyses.
Use has been made of the mini-cell system to study polypeptide synthesis from cloned EcoRI, HindIII and PstI fragments of T5 DNA. The correlation of certain gene products with known genes has been established, as well as the physical mapping of genes not yet identified genetically. In some cases, it has been possible to demonstrate the presence of T5 promoters on the cloned DNA fragments. The design of experiments to avoid certain artifacts inherent in the use of the mini-cell system is discussed.
We have analyzed the kinetoplast DNA for Trypanosoma equiperdum (American Type Culture Collection 30019) and two dyskinetoplastic strains derived from it. The DNA networks from the kinetoplastic strain are made up of catenated mini-circles and maxi-circles, like the networks from the closely-related Trypanosoma brucei. The mini-circles of T. equiperdum lack the pronounced sequence heterogeneity of T. brucei mini-circles, as shown by the fragment distribution of restriction digests and by the predominance of well-matched duplexes in electron micrographs of renatured DNA. The electrophoretic analysis of kinetoplast DNA digested with various restriction endonucleases shows the maxi-circle of T. equiperdum to consist of circular DNA molecules of 8.4 x 10(6) daltons, without size or sequence heterogeneity or repetitious segments. A comparison of the sequence by restriction endonuclease fragmentation and hybridization shows extensive sequence homology. The size difference between both maxi-circles is due to the deletion of one continuous segment of 5.10(6) daltons. In the two dyskinetoplastic strains, we cannot detect DNA sequences that hybridize with kinetoplast DNA from T. brucei or from the kinetoplastic strain of T. equiperdum. In one of these strains, a 'low-density' DNA fraction contained a simple sequence DNA, cleaved by restriction endonuclease HindIII into fragments of 180 base-pairs and multimers of this. The relation of this DNA to kinetoplast DNA, if any, is unknown.
Maxi-circles are a minor component of kinetoplast DNAs from all trypanosomatids studied, but they have not previously been found in Trypanosoma cruzi; We have spread intact kinetoplast DNA from the epimastigotes of strain Y in protein monolayers and analysed the mini-circle networks by electron microscopy. Long loops up to 10 micrometer were present, extending from the network rim; these are considered typical of maxi-circles. The presence of maxi-circles was proven by digestion of kinetoplast DNA with restriction endonucleases and S1 nuclease. This released a minor DNA component, detectable by agarose gel electrophoresis, which hybridized to maxi-circle DNA from Trypanosoma brucei. The molecular weight of the linearized maxi-circle of Trypanosoma cruzi is 26 . 10(6), as judged from its electrophoretic mobility in 0.6% agarose. Our restriction enzyme analysis of the mini-circles of Trypanosoma cruzi has confirmed their sequence heterogeneity and internally-repeated structure. We have found that more than 90% of the mini-circles are cut into 1/4 length molecules by endonuclease TaqI. Denaturation and renaturation of mini-circles, cut once with endonuclease MboI, mainly yields linear and circular molecules with single-stranded eyes and tails in electron micrographs. This shows that 1/4 repeats contain sub-segments in which sequence divergence is extensive. Our EcoRI and HapII digests differ in fragment size distribution from those previously reported. This suggests that this distribution may not be a stable characteristic of the Y strain.