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Biomedical subjects

B Kemper

Publications and source records attributed to B Kemper.

At least 127 records · Page 7Linked to original sources
PubMed1984

Isolation and sequence analysis of three cloned cDNAs for rabbit liver proteins that are related to rabbit cytochrome P-450 (form 2), the major phenobarbital-inducible form.

We have isolated from rabbit liver three cDNA clones of 1400-1800 base pairs that hybridize selectively to RNA from animals treated with phenobarbital. The nucleotide sequences of the cDNAs have been determined. In the protein coding region the nucleotide sequences of two of the cDNAs are 88% homologous, and the third cDNA is about 72-74% homologous to the other two. All three are 55-60% homologous to rat liver cytochrome P-450b cDNA. The amino acid sequences derived from the cDNA sequences are about 50% homologous to those of rat liver cytochrome P-450b and rabbit liver cytochrome P-450 (form 2). The degree of homology differs substantially in different regions of the protein. The hydrophobicity profiles of these five mammalian cytochromes P-450 are very similar and contain up to eight regions of hydrophobicity that are long enough to span a membrane. These results indicate that these three cDNAs code for rabbit liver cytochromes P-450 which are different from any rabbit liver cytochrome P-450 for which amino acid sequence information is published. These cDNAs are part of a family of genes that are related to rabbit liver cytochrome P-450 (form 2) and rat liver cytochrome P-450b which are the major phenobarbital-inducible forms. The divergence of amino acid sequence between the rat and rabbit forms and the divergence of nucleotide sequences of silent sites in the two most closely related rabbit forms suggest that cytochromes P-450 have a relatively high rate of amino acid divergence compared to many other vertebrate proteins.

Amino Acid Sequence↗
PubMed1984

The compleat otolaryngologist: Burton Alexander Randall.

The practicing otolaryngologist and resident is the beneficiary of a highly organized educational system which has evolved over the past 100 years. A number of social, scientific and technical advances were involved in the evolution of these training programs; however, one of the most important developments was the emergence of the Clinician Educator Concept. Burton Alexander Randall was prominent in the profession in the late nineteenth century and early twentieth century and is an example of the clinician-teacher who participated in the development of otorhinolaryngologic instruction. His unpublished memoirs, recently made available, illustrate the difficulties in obtaining appropriate training, organizing clinical services, and establishing professional relationships. These interesting documents provide insight into the present and illustrate that although there have been great technical advances, some problems in interpersonal relationships remain the same.

Education, Medical↗
PubMed1984

Cruciform-resolvase interactions in supercoiled DNA.

T4 endonuclease VII, which cleaves Holliday-like junctions in DNA, specifically cleaves short inverted repeats in supercoiled plasmids. These sequences are subject to site-specific cleavage by single-strand-specific nucleases, and cruciform formation has been suggested as an explanation for this observation. This proposal is greatly strengthened by the present data, since a formal analogy between cruciform structures and Holliday junctions exists. Resolution of a variety of unrelated cruciform sequences demonstrates that the cleavage process results in a linear molecule with hairpin ends and single ligatable nicks at positions corresponding to the stem-base of the cruciform. In two examples mapped in detail, the cleavages are exclusively introduced at two or three nucleotides from the end of the symmetric sequence at the 5' side on each strand. These studies demonstrate the potential of endonuclease VII as a probe of cruciform structure and the utility of short cruciform structures as Holliday junction models.

Base Sequence↗
PubMed1984

Isolation and complete nucleotide sequence of the gene for bovine parathyroid hormone.

The structure of the bovine parathyroid hormone (PTH) gene has been analyzed by Southern blot hybridization of genomic DNA and by nucleotide sequence analysis of a cloned PTH gene. In the Southern analysis, several restriction enzymes produced single fragments that hybridized to PTH cDNA suggesting that there is a single bovine PTH gene. The restriction map of the cloned gene is the same as that determined by Southern blot analysis of bovine DNA. The sequence of 3154 bp of the cloned gene has been determined including 510 bp and 139 bp in the 5' and 3' flanking regions, respectively. The gene contains two introns which separate three exons that code primarily for: (i) the 5' untranslated region, (ii) the pre-sequence of preProPTH, and (iii) PTH and the 3' untranslated region. The gene contains 68% A + T and unusually long stretches of 100- to 150-bp sequences containing alternating A and T nucleotides in the 5' flanking region and intron A. The 5' flanking region contains two TATA sequences, both of which appear to be functional as determined by S1 nuclease mapping. Compared to the rat and human genes, the locations of the introns are identical but the sizes differ. Comparable human and bovine sequences in the flanking regions and introns are about 80% homologous.

Animals↗
PubMed1983

Excision of amplified viral DNA at palindromic sequences from the adenovirus type 12-transformed hamster cell line T637.

In the DNA of the adenovirus type 12 (Ad12)-transformed hamster cell line T637 approximately 20-22 viral DNA molecules per cell are covalently linked to cellular DNA. Spontaneously arising morphological revertants of T637 cells have lost the bulk of the viral DNA. We have been able to mimic the excision event of viral DNA, as it occurs during reversion, by autoincubation of isolated nuclei from T637 cells. The same Ad12 DNA sequences, which had been deleted in morphological revertants, proved highly sensitive to endogenous nucleases in isolated nuclei of T637 cells. Viral DNA sequences, which persisted in the revertants, are resistant to endogenous nucleases in isolated T637 nuclei. All attempts to clone the nuclease-sensitive sites of Ad12 DNA in cell line T637 have so far failed. After denaturation and renaturation of T637 DNA followed by treatment with S1 nuclease, large fold-back structures of DNA have been found. These snap-back structures were derived from precisely those viral DNA restriction fragments which were uncloneable. The fragments containing palindromic sequences were both highly sensitive to endogenous nucleases in isolated T637 nuclei and were absent from the DNA of all revertant cell lines. Moreover, the palindromic sequences are susceptible to the phage T4-specific endonuclease VII which specifically attacks cruciform structures in DNA. The peculiar structures at the termini of integrated Ad12 DNA molecules are highly sensitive to endogenous nucleases in isolated nuclei. These nucleases may be related to the reversion event.

Adenoviruses, Human↗
PubMed1982

T4 endonuclease VII cleaves holliday structures.

T4 endonuclease VII cleaves Holliday structures in vitro by cutting two strands of the same polarity at or near the branch point. The two unbranched duplexes produced by cleavage each contain a strand break that can be sealed by DNA ligase. This suggests that the cut sites are at the same position in the nucleotide sequence in each strand. The joint action of endonuclease VII and DNA ligase can therefore resolve Holliday structures into genetically sensible products. These observations account for the role of endonuclease VII in the DNA metabolism of phage T4, and provide the first example of an enzyme that acts specifically on branch points in duplex DNA.

Bacteriophage lambda↗
PubMed1981

Studies on T4-head maturation. 1. Purification and characterization of gene-49-controlled endonuclease.

An endonuclease has been purified more than 300-fold from Escherichia coli infected with bacteriophage T4. The enzyme degrades rapidly sedimenting (greater than 1000 S) DNA in vitro by introducing a limited number of breaks. The substrate is the replicative DNA isolated from cells infected with gene-49-defective phage [Kemper, B, and Janz, E. (1976) J. Virol. 18, 992-999]. Molecules of approximately a third the size of unit-length T4 DNA are exclusively found in a limit digest. The enzyme also reacts with single-stranded DNA from various sources. Heat-denatured T4 DNA is converted into acid-soluble oligonucleotides. Circular single-stranded M13 DNA is linearized by endonucleolytic cleavage causing a reduction of infectivity during transfection. The enzyme behaves like a typical late-gene product. Its activity is 100-fold reduced in cells infected with gene-55-defective phage (defect in expression of late functions). A 30-fold reduction in its specific activity was found in cells infected with gene-49-defective phage suggesting that gene 49 codes for the enzyme or controls its expression. The purified enzyme binds to native or denatured DNA from various sources. The protein has a molecular weight of 42000 as determined by gel filtration and sedimentation analysis. Optimal activity on rapidly sedimenting DNA is obtained at pH 8.6 in Tris/HCl buffer in the presence of 10 mM MgCl2. Some 75% of the activity can be obtained with 7 mM MnCl2. 5 mM CaCl2 has a stimulatory effect on the reaction with MgCl2 or MnCl2 each present at its individual optimal concentration. The enzyme does not require the addition of sulfhydryl reagent for full activity. The reaction can be inhibited by compounds like KCl, spermidine, p-hydroxymercuribenzoate or tRNA.

Chemical Phenomena↗
PubMed1981

Studies on T4-head maturation. 2. Substrate specificity of gene-49-controlled endonuclease.

The substrate specificity of 49+-enzyme was investigated in vitro. The enzyme showed a marked preference for rapidly sedimenting T4 DNA (greater than 1000 S) when helix-destabilizing proteins from Escherichia coli or phage T4 were added to the reaction. Regular replicative T4 DNA (200-S DNA) or denatured T4 DNA was not cleaved by the enzyme in the presence of these proteins but if they were omitted from the reaction both DNAs become good substrates for the enzyme. 200-S DNA was cleaved at its natural sites of single strandedness which occur at one-genome intervals. Gaps in T4 DNA which were constructed by treatment of a nicked DNA with exonuclease III were also cleaved by 49+-enzyme in the absence of helix-destabilizing proteins. Single-stranded T4 DNA was extensively degraded and up to 50% of the material was found to be acid-soluble in a limit digest. The degradation products were predominantly oligonucleotides of random size. No preference for a 5'-terminal nucleotide was observed in material from a limit digest with M13 DNA. Double-stranded DNA was nicked upon exposure to 49+-enzyme and double-strand breakage finally occurred by an accumulation of single-strand interruptions. No acid-soluble material was produced from native T4 DNA. The introduction of nicks in native DNA did not improve its properties as a substrate for the enzyme. Double-stranded DNA was about 100-fold less sensitive to the enzyme than single-stranded DNA.

DNA, Viral↗
PubMed1981

Introduction by molecular cloning of artifactual inverted sequences at the 5' terminus of the sense strand of bovine parathyroid hormone cDNA.

To study the structure and function of the gene for parathyroid hormone, we obtained recombinant plasmids containing bovine parathyroid hormone cDNA. The nucleotide sequence at the 5' terminus (relative to the sense strand) of the cDNA insert in a recombinant plasmid, pPTHi4, was different from that previously reported for the bovine parathyroid hormone cDNA insert of another recombinant plasmid, pPTHm1 [Kronenberg, H. M., McDevitt, B. F., Majzoub, J. A., Nathans, J., Sharp, P. A., Potts, J. T., Jr. & Rich, A. (1979) Proc. Natl. Acad. Sci. USA 76, 4981-4985]. The first 50 nucleotides of the pPTHm1 insert were an inverted complement of nucleotides 2-51 of the pPTHi4 insert. the cDNA insert of another plasmid, pPTHi8, contained a sequence identical to nucleotides 2-51 of the pPTHi4 insert but also contained an additional 42 bases at the 5' terminus. The first 41 bases of the pPTHi8 insert were an inverted repeat of an internal sequence of the pPTHi4 insert corresponding to nucleotides 184-224. Restriction endonuclease analysis of pPTHi8 indicated that the internal sequence corresponding to this region was retained. The nucleotide sequence of a restriction fragment hybridized to parathyroid hormone mRNA and extended toward the 5' terminus of the mRNA with reverse transcriptase confirmed that the sequence at the 5' terminus of the pPTHi4 insert was an accurate copy of the parathyroid hormone mRNA sequence. These data suggest that two types of sequence rearrangements may occur at the 5' terminus, as occurred in pPTHm1, and (ii) an inverted repeat of an internal sequence, as occurred in pPTHi8.

Animals↗
PubMed1980

Synthesis, restriction analysis, and molecular cloning of near full length DNA complementary to bovine parathyroid hormone mRNA.

DNA complementary (cDNA) to a partially purified preparation of bovine parathyroid hormone mRNA was synthesized using avian myeloblastosis viral reverse transcriptase. The PTH cDNA contained about 750 bases and was greater than 95% sensitive to digestion by S1 nuclease. Analysis of the mRNA preparation by excess RNA hybridization to the PTH cDNA revealed one rapidly hybridizing component consisting of 50% of the PTH cDNA. Sequential incubation of the PTH mRNA with reverse transcriptase and E. coli DNA polymerase I produced near full length double-stranded PTH cDNA. Of the 22 restriction endonucleases tested, double-stranded PTH cDNA could be cleaved with Alu I, Mbo II, Sau 3A, Sst I, and Taq I. The restriction fragments corresponding to the 5' terminus of the sense strand were identified for the last three enzymes by comparing the size of fragments obtained from PTH cDNA before and after cleavage of the hairpin loop connecting the two strands by S1 nuclease. The restriction map of the cDNA was used to detect clones of bacteria containing recombinant plasmids with near full length PTH cDNA inserts.

Animals↗
PubMed1978

Pre-proparathyroid hormone; amino acid sequence, chemical synthesis, and some biological studies of the precursor region.

The precursor of bovine proparathyroid hormone was synthesized by translation of parathyroid mRNA in a wheat-germ cell-free system. The amino acid sequence of the NH2-terminal extension (the pre sequence) was determined by repetitive Edman degradation of the polypeptide labeled with radioactive amino acids (radiosequencing). The pre sequence of pre-proparathyroid hormone is (formula: see text) which is followed by the sequence of proparathyroid hormone. It is significant that 20 of the 25 amino acids in the sequence are hydrophobic. This high hydrophobicity is consistent with the proposed role of the pre sequence as a membrane-penetrating peptide. The precursor-specific sequence of 31 amino acids was snythesized chemically by the solid-phase technique. This synthetic peptide was shown to bind to the microsomal fraction of homogenates prepared from extracts of parathyroid glands, a finding consistent with the proposed role of the precursor peptide in the attachment of the nascent chain--mRNA--ribosome complex to the endoplasmic reticulum.

Amino Acid Sequence↗