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Biomedical subjects

R K Patient

Publications and source records attributed to R K Patient.

44 records · Page 3Linked to original sources
PubMed1980

Internal organization of the major adult alpha- and beta-globin genes of X. laevis.

We describe the isolation of two recombinant lambda phages, each containing genomic DNA fragments encoding both the major adult alpha- and beta-globin mRNAs of X. laevis. The DNA fragment in the two clones have restriction maps which indicate that they are each derived from a different member of the pair of alleles present in the heterozygote used as the source of DNA for cloning. The characterization of these two clones by restriction mapping, R looping and DNA sequencing shows that the alpha 1- and beta 1-globin genes lie in the orientation separated by 7.7 kb of DNA. There are two introns in the alpha 1-globin gene and two in the beta 1-globin gene, and they interrupt the genes at exactly the same positions as the introns found in all known mammalian alpha- and beta-globin genes. The exon sequences proximal to the introns show a much higher degree of homology with mammalian sequences than the sequences distal to intron/exon junctions, and the introns in the beta 1-globin gene of X. laevis are very similar in length to the corresponding introns in the beta-globin genes of several mammals and the chicken.

Animals↗
PubMed1979

Characterization of in vitro transcription initiation and termination sites in Col E1 DNA.

Overlapping restriction fragments from the region between the single Eco R1 site and the origin of replication of the plasmid, Col E1, have been utilised as templates in an in vitro transcription assay using E. coli RNA polymerase. Transcription towards the single Eco R1 site is initiated at a point 415 bp to the origin side of that site. In vivo, transcription starting at this point probably produces the mRNA for the colicin immunity protein. Transcription away from the Eco R1 site is initiated at a point 140 bp to the origin side of that site and terminated 30 bp further on. This terminator is probably the point at which transcription of the colicin gene is terminated in vivo. DNA sequence analysis in both these regions demonstrated several similarities to other prokaryotic regulatory regions. 50% homology between the putative immunity promoter and other prokaryotic promoters is apparent, so are similarities in AT-content. Upstream of the ATG start codon the sequence PuPuTTTPuPu and a termination codon (TAA) appear; both are typical of prokaryotic ribosome binding sites. The colicin terminator demonstrated similarities to other rho-independent prokaryotic terminators: a GC-rich region with termination in an adjacent AT-rich region containing T clusters on the non-coding strand. The possible role of initiation upstream from the colicin terminator is discussed.

Base Sequence↗
PubMed1979

Construction and mapping of recombinant plasmids used for the preparation of DNA fragments containing the Escherichia coli lactose operator and promoter.

Three DNA restriction fragments of established sequence containing the Escherichia coli lac genetic controlling regions were cloned. In each case a recombinant plasmid was constructed which was suitable for the subsequent large scale purification of the lac fragment. A 789-base pair HindII fragment, containing the lac operator, promoter, and cyclic AMP receptor protein binding site, was ligated into the single HindII site of the amplifiable plasmid minicolicin E1 DNA (pVH51). A 203-base pair Hae III fragment containing the same genetic sites was ligated into the single Eco RI site of pVH51 which had been "filled in" by the Micrococcus luteus DNA polymerase. Thus, the lac fragment was inserted between two Eco RI sites. Plasmids containing multiple copies of this Eco RI fragment were then constructed. A 95-base pair Alu I fragment containing the lac promoter and operator was cloned similarly. Also, the 203-base pair fragment was cloned into the Eco RI site of pVH51 using a 300-base pair linker fragment (isolated by RPC-5 column chromatography) which permitted retention of its Hae III ends. Mapping studies on pVH51 DNA with a number of DNA restriction endonucleases, including Alu I, Taq I, and Hpa II, are described.

DNA Restriction Enzymes↗
PubMed1979

Identification of regions of pRZ2 which have delayed elution behavior on RPC-5 column chromatography.

DNA restriction fragments generally elute from RPC-5 in order of their size. However, some fragments elute later than predicted. Chromatographic studies were performed on five different restriction digests (Hae III, Hha I, Alu I, Taq I, and Hae III + HindII) of pRZ2 DNA in an effort to localize the regions which have the delayed properties. Also, the magnitude of the delay was quantitated in each case. Most of the delayed fragments were localized in one major (931 bp) and one minor (approximately 210 bp) region of the genome. The fragments exhibiting a greater extent of delay were in the major region. The results described herein and in the following paper show that, in most cases, this effect can be explained by the base composition, or sequence of the fragments, or both.

Chromatography, High Pressure Liquid↗
PubMed1979

Influence of A-T content on the fractionation of DNA restriction fragments by RPC-5 column chromatography.

The properties of the fractionated Hae III fragments of pRZ2 DNA (Patient, R.K., Hardies, S.C., and Wells, R.D. (1979) J. Biol. Chem. 254, 5542-5547) were studied in an effort to determine why several of the fragments bind more tightly to RPC-5 than expected on the basis of their length. The purified fragments were analyzed for their nucleotide composition by direct determination of their constituent mononucleotides and by analytical CsCl and Cs2SO4 density gradient analyses. A-T-rich fragments elute at higher salt concentrations than fragments of equivalent size which are not A-T-rich. In addition, denaturation mapping studies by electron microscopy indicate that an A-T-rich run within an otherwise G-C-rich fragment can give rise to delayed elution. At least one other factor influences the separation of DNA restriction fragments by RPC-5 chromatography. Some of the fragments in this digest which elute later than predicted from their size either contain known genetic regulatory sites or bind regulatory proteins.

Adenine↗
PubMedDate not supplied

Simian virus 40-mediated cis induction of the Xenopus beta-globin DNase I hypersensitive site.

Regions in chromatin which are hypersensitive to the action of DNase I appear to be associated with sites of genetic activity; the association between DNase I hypersensitivity and transcriptional activation is well known. In the case of the chicken beta-globin gene the establishment of a DNase I hypersensitive site is dependent on tissue-specific trans-acting factors. Such factors have also been implicated in the action of viral and cellular enhancers, which are themselves hypersensitive to DNase I. Enhancers have been defined operationally as DNA sequences which act in cis to potentiate transcription from their own, heterologous or cryptic promoters. This activity is essentially unaffected by changes in the orientation, position (5' or 3') or distance of the enhancer element with respect to its cognate promoter. We demonstrate here that the transcriptional rescue of the Xenopus laevis beta-globin gene by simian virus 40 (SV40) sequences including the enhancer coincides with the conferment of DNase I hypersensitivity upon that gene, and that this occurs in the absence of any change in the complement of trans-acting factors. These results suggest that a propensity to form sites hypersensitive to the action of DNase I is encoded in the primary sequence of DNA, and that this predilection is aggravated by SV40 sequences, perhaps through a mechanism dependent on supercoiling.

Animals↗