Alan D. B. Malcolm

Differential reactivities at restriction enzyme sites

A method has been developed to measure the rates of digestion by restriction enzymes at individual sites. This involves a simple arithmetical treatment of the integrated areas from a densitometer scan of an ethidium bromide stained gel. We have used this method to study the digestion by HpaI, HincII and SalI of pBR322 and phi X174 DNA, and the effect of various DNA binding ligands. One of the two HpaI sites in phi X174 DNA is much more sensitive to inhibition by ligands such as netropsin, which display a preference for AT base pairs, than is the other site. Inspection of the sequences flanking the restriction sites shows that the former contains a much higher proportion of AT base-pairs than dose the latter. The opposite phenomenon is observed with the two HincII sites in pBR322. This illustrates the importance of neighbouring sequences in the interaction between restriction enzymes and their cleavage sites in DNA.

Effects of ppGpp on transcription by DNA-dependent RNA polymerase from Escherichia coli: circular dichroism, absorption and specific transcription studies

Concrete evidence is presented for conformational changes elicited in RNA polymerase upon binding ppGpp by circular dichroism measurements. In the presence of 100 microM ppGpp, the molar ellipticity of RNA polymerase at 220 nm is reduced by 14% from the initial value of - 11,100 deg X cm2 X dmol-1 at 25 degrees C. In vitro transcription on templates containing the beta-lactamase promoter and colicin E1 promoter on poly[d(A-T)] is inhibited by ppGpp. None of these templates had GC-rich nucleotide sequence near the transcription initiation site, and yet they were influenced by ppGpp. Comparison of the effect on the synthesis of mRNAs for beta-lactamase and colicin E1 and the synthesis of the proteins themselves indicates that the effect of ppGpp is at the level of transcription for the former case and involves coupled transcription-translation for the latter case. Difference absorption, polyacrylamide gel electrophoresis, and nitrocellulose filter-binding studies show that the binding of ppGpp to RNA polymerase does not impair the extent of the interaction between enzyme and DNA. Kinetic studies suggest that ppGpp affects transcription initiation on beta-lactamase promoter. On poly[d(A-T)], ppGpp affects the rate of open complex formation and is a mixed inhibitor with respect to the incorporation of nucleotides. Our results are consistent with the idea that ppGpp acts as a regulator by binding at a site different from the active site and changes the RNA polymerase conformation, causing altered transcriptional behavior on different DNA templates.

Cross-hybridisation of human papillomavirus DNA on filters

Accurate type assignment of the different HPV types which infect the female genital tract, is essential in view of the differing pathological potential of the common virus types present in the cervix. We have developed hybridisation, washing and autoradiography conditions that minimise cross-hybridisation on filters and so allow clear-cut type assignment. We describe the conditions in this paper and have used this method to screen for HPV infection in clinical populations.

Differential inhibition of a restriction enzyme by quinoxaline antibiotics

The inhibition of cleavage by HpaI at two well-defined restriction sites in linearised phi X174-RF DNA by quinoxaline antibiotics has been investigated. Echinomycin, which displays a certain preference for binding to GC basepairs, inhibits cleavage at one site much more than the other, whereas triostin A, which displays less pronounced sequence-selectivity, inhibits both sites about equally. Other congeners inhibit reaction at the two sites with varying effectiveness. The results demonstrate the usefulness of studying inhibition of cleavage at specific sites by restriction enzymes as a means of exploring the specificity of DNA-ligand interactions.

Preparative Scale, High Resolution Purification of Low Molecular Weight DNA Fragments

A commercially available continuous electroelution system has been used to separate and purify low molecular weight DNA fragments from polyacrylamide gels. This method has several advantages over previously reported methods for the recovery of DNA fragments from polyacrylamide gels. This technique, which gives very high recovery rates (80-95%), can be carried out on a relatively large scale and in a way that is not labour intensive. Data are presented for the purification of DNA fragments with molecular weights in the range 1-4 x 10(5) (200-700 base-pairs), although the method is also applicable to larger molecular weight DNA fragments, RNAs and proteins.

Dna Hybridization of Cervical Tissues

The increasing frequency of cervical neoplasia among younger women and the increased invasiveness of these tumors has led to a considerable growth in research into this disease. Conventional methods (epidemiology, cytology, and immunology), while being extremely useful, also have significant limitations. Recent advances in techniques for the manipulation of DNA now make it possible to analyze tissues for the presence of viral genomes. This review introduces these techniques and describes their application to the search for herpes simplex virus and human papillomavirus sequences in cervical tissue. The significance of the findings both for the mechanism of transmission of the disease, and also the consequences for early detection and hence more successful treatment, are also discussed.

Restriction Enzymes and DNA

The discovery of restriction and modification enzymes, which proved to be a major turning point in the progress of molecular biology, was a consequence of a bacteriological observation in the early 1950s (Luria and Human, 1952; Bertani and Weigle, 1953). The two groups reported the curious behaviour of phage grown on two different strains of bacteria. Phages propagated on one strain were found to grow poorly on the second (hence the term ‘restriction’) and vice versa. However, the few phages that escaped restriction could then grow well on the new host, thus being modified in a way that afforded them protection from the restriction imposed by the host.