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Biomedical subjects

A R Oliphant

Publications and source records attributed to A R Oliphant.

9 recordsLinked to original sources
PubMed1990

Identification of mutations in the COL4A5 collagen gene in Alport syndrome.

X-linked Alport syndrome is a hereditary glomerulonephritis in which progressive loss of kidney function is often accompanied by progressive loss of hearing. Ultrastructural defects in glomerular basement membranes (GBM) of Alport syndrome patients implicate an altered structural protein as the cause of nephritis. The product of COL4A5, the alpha 5(IV) collagen chain, is a specific component of GBM within the kidney, and the gene maps to the same X chromosomal region as does Alport syndrome. Three structural aberrations were found in COL4A5, in intragenic deletion, a Pst I site variant, and an uncharacterized abnormality, which appear to cause nephritis and deafness, with allele-specific severity, in three Alport syndrome kindreds in Utah.

Blotting, Southern↗
PubMed1989

An efficient method for generating proteins with altered enzymatic properties: application to beta-lactamase.

Random-sequence or highly degenerate oligonucleotides have been useful for defining functionally important sequences both in proteins and in nucleic acids. In this approach, such oligonucleotides are used to replace a segment of DNA required for a desired function, and functional sequences are identified by an appropriate genetic or biochemical selection. Here, a collection of 500,000 [corrected] altered beta-lactamase proteins was generated by cloning a mixed-base oligonucleotide in place of the sequences coding for a 17-amino acid portion of the enzyme's active site. Approximately 2000 enzymes from this collection were able to confer ampicillin resistance on Escherichia coli. Fifty-eight of these were chosen for further study after characterization with various beta-lactam substrates. beta-Lactamases having altered specificity against different antibiotics, resistance to the suicide inhibitors clavulanic acid and sulbactam, and temperature-dependent activities were obtained. The amino acid residues responsible for these altered properties as well as for basic enzyme activity are defined. This approach should prove to be an effective and general tool for creating proteins with novel properties, especially in situations in which a high-resolution structure of the protein is not known.

Amino Acid Sequence↗
PubMed1989

Defining the sequence specificity of DNA-binding proteins by selecting binding sites from random-sequence oligonucleotides: analysis of yeast GCN4 protein.

We describe a new method for accurately defining the sequence recognition properties of DNA-binding proteins by selecting high-affinity binding sites from random-sequence DNA. The yeast transcriptional activator protein GCN4 was coupled to a Sepharose column, and binding sites were isolated by passing short, random-sequence oligonucleotides over the column and eluting them with increasing salt concentrations. Of 43 specifically bound oligonucleotides, 40 contained the symmetric sequence TGA(C/G)TCA, whereas the other 3 contained sequences matching six of these seven bases. The extreme preference for this 7-base-pair sequence suggests that each position directly contacts GCN4. The three nucleotide positions on each side of this core heptanucleotide also showed sequence preferences, indicating their effect on GCN4 binding. Interestingly, deviations in the core and a stronger sequence preference in the flanking region were found on one side of the central C . G base pair. Although GCN4 binds as a dimer, this asymmetry supports a model in which interactions on each side of the binding site are not equivalent. The random selection method should prove generally useful for defining the specificities of other DNA-binding proteins and for identifying putative target sequences from genomic DNA.

Base Sequence↗
PubMed1988

Defining the consensus sequences of E.coli promoter elements by random selection.

The consensus sequence of E.coli promoter elements was determined by the method of random selection. A large collection of hybrid molecules was produced in which random-sequence oligonucleotides were cloned in place of a wild-type promoter element, and functional -10 and -35 E.coli promoter elements were obtained by a genetic selection involving the expression of a structural gene. The DNA sequences and relative levels of function for -10 and -35 elements were determined. The consensus sequences determined by this approach are very similar to those determined by comparing DNA sequences of naturally occurring E.coli promoters. However, no strong correlation is observed between similarity to the consensus and relative level of function. The results are considered in terms of E.coli promoter function and of the general applicability of the random selection method.

Base Sequence↗
PubMed1986

Cloning of random-sequence oligodeoxynucleotides.

Methods are described for cloning random or highly degenerate nucleotide (nt) sequences. The procedures use synthetically derived mixtures of oligodeoxynucleotides (oligos) whose heterogeneous central portions are bounded at their 5' and 3' ends by sequences recognized by restriction endonucleases. Oligo collections of defined length and nt composition are synthesized by utilizing appropriate concentrations of all four nucleotide precursors during each addition step for the central region. Single-stranded oligos with appropriate 5' and 3' ends can be ligated directly, although inefficiently, into double-stranded (ds) DNA molecules with complementary 5' and 3' extensions produced by restriction endonuclease cleavage. A more general and efficient method is to convert the oligo into a ds form by incubating it with the Klenow (large) fragment of Escherichia coli DNA polymerase I. If the 3' ends are palindromic, two oligo molecules will serve as mutual primers for polymerization. The resulting products are ds molecules containing two oligo units separated by the original 3' restriction site and bounded at each end by the original 5' restriction site. After appropriate restriction endonuclease cleavage, oligo units can be cloned by standard procedures. Analysis of 26 recombinant M13 phages indicates that the nt sequences of the cloned oligos are in good accord with what was expected on a random basis.

Base Sequence↗