PubMed Health⌕ Search

Biomedical subjects

B Kemper

Publications and source records attributed to B Kemper.

At least 109 records · Page 6Linked to original sources
PubMed1989

N-terminal basic amino acids are not required for translocation and processing of preproparathyroid hormone.

An N-terminal deletion mutant of preproparathyroid hormone that contains a single basic amino acid, lysine, in the N-terminal domain of the signal peptide is translocated across the endoplasmic reticulum membrane similarly to intact preproparathyroid hormone. To examine the function of charged residues preceeding the hydrophobic core, the lysine was replaced by an uncharged (methionine) or negatively charged (glutamic acid) amino acid. The translocational activity of the mutant signal peptides was assayed in a reticulocyte lysate system containing chicken oviduct microsomal membranes. Altering the net charge of the N-terminal domain did not abolish signal sequence activity, although the efficiency of translocation was decreased for the mutant with a glutamic acid substitution. Posttranslational, ribosome independent, translocation was observed for all the mutants tested, with the same dependence on N-terminal charge but with much lower efficiency than cotranslational translocation. These studies show that the presence of basic amino acids in the N-terminal domain of a eukaryotic signal sequence is not required for its activity.

Amino Acid Sequence↗
PubMed1988

The structure of the Holliday junction, and its resolution.

The Holliday (four-way) junction is a critical intermediate in homologous genetic recombination. We have studied the structure of a series of four-way junctions, constructed by hybridization of four 80 nucleotide synthetic oligonucleotides. These molecules migrate anomalously slowly in gel electrophoresis. Each arm of any junction could be selectively shortened by cleavage at a unique restriction site, and we have studied the relative gel mobilities of species in which two arms were cleaved. The pattern of fragments observed argues strongly for a structure with two-fold symmetry, based on an X shape, the long arms of which are made from pairwise colinear association of helical arms. The choice of partners is governed by the base sequence at the junction, allowing a potential isomerization between equivalent structural forms. Resolvase enzymes can distinguish between these structures, and the resolution products are determined by the structure adopted, i.e., by the sequence at the junction. In the absence of cations, the helical arms of the junction are fully extended in a square configuration, and unstacking results in junction thymines becoming reactive to osmium tetroxide.

Base Sequence↗
PubMed1988

Initiation of heteroduplex-loop repair by T4-encoded endonuclease VII in vitro.

Heteroduplex DNAs with single-stranded loops of 51 nt or 8 nt were constructed in vitro and used in reactions with purified endonuclease VII (endo VII) from phage T4. The enzyme makes double-strand breaks by introducing pairs of staggered nicks flanking the loops. Regardless of loop-size the nicking sites map exclusively at the 3' side of the loop in the looping strand and at the 3' side of the base of the loop in the non-looping strand. The number of potential cleavage sites is small (less than 5) and their distribution depends on DNA sequence. The two closest staggered nicks are 4 bp apart, 2 bp on either side of the loop. Nicking always occurs in the double-stranded part of the molecules; the single-stranded loops are not attacked by endo VII. The nicks are introduced in a stepwise fashion and selection of the strand for the first nick depends on the sequence of 31 base pairs flanking the loops.

Base Sequence↗
PubMed1988

Chimeric single-stranded DNA phage-plasmid cloning vectors.

A variety of ssDNA phagemid cloning vectors have been constructed that combine the advantages of the filamentous coliphages with a number of plasmid and other bacteriophage-encoded functions. The practical and biological advantages of a chimeric phage-plasmid vector are considerable, and the trend toward converting existing plasmids into ssDNA phagemids and consolidating any number of useful features into one or a few vectors will undoubtedly accelerate. A new helper phage specifically designed for the isolation of large amounts of single-stranded plasmid DNA simplifies the use of these cloning vehicles. Additional refinements in phagemid-helper phage systems should extend the potential of these vectors even further.

Bacteriophages↗
PubMed1988

T4 endonuclease VII cleaves the crossover strands of Holliday junction analogs.

We have formed four-arm branched DNA junctions that contain no more than a single base pair of branch migratory freedom. Recently, we have shown that these Holliday junction analogs have twofold symmetric protection patterns in solution when probed with hydroxyl radicals: two opposite strands of one junction show extensive protection near the branch point, while the other pair of opposite strands is virtually as susceptible as a double helix. In a different junction, the hydroxyl radical protection pattern is reversed. These patterns suggest that a crossover-isomer bias exists in these molecules and that the protected strands form the crossover between helices. Here, we examine the cleavage pattern of these structures when they are resolved by T4 endonuclease VII. Junctions are formed from a single shamrock-shaped molecule, which contains 5', 3', or internal labels. The enzyme shows a preference for resolving these modified junctions at sites near those protected from hydroxyl radicals. This result suggests that only crossover strands in a Holliday junction are cleaved, and thus an odd number of crossover isomerizations must occur when flanking markers are exchanged.

Base Sequence↗
PubMed1988

Positive charges at the NH2 terminus convert the membrane-anchor signal peptide of cytochrome P-450 to a secretory signal peptide.

The NH2-terminal sequences of cytochromes P-450 resemble signal peptides, but these sequences are not cleaved during the insertion of these integral membrane proteins into the microsomes. To examine whether these putative signal peptides are functionally equivalent to signal peptides of secretory proteins, cDNA coding for a fusion protein was produced, in which the signal peptide for preproparathyroid hormone was replaced with the putative signal peptide of cytochrome P450IIC2. The translational product of RNA synthesized in vitro from the cDNA was neither processed nor translocated by chicken oviduct microsomal membranes in a reticulocyte cell-free system but was resistant to extraction from the membranes by alkaline solutions. In addition, the translation of the hybrid RNA was arrested by signal recognition particle. Unlike most signal peptides, the cytochrome P450IIC2 NH2-terminal sequence does not contain basic amino acids preceding the hydrophobic core. Introduction by oligonucleotide-directed mutagenesis of lysine and arginine at the NH2 terminus resulted in a fusion protein that was partially processed by the microsomal membranes, with translocation across the membrane of both the processed and unprocessed proteins. The positive charges convert the cytochrome P450IIC2 NH2 terminus from a combination membrane insertion-halt transfer signal to a more classical secretory membrane-insertion signal, possibly by altering the orientation of the signal peptide in the membrane.

Amino Acid Sequence↗
PubMed1987

Characterization of rabbit cytochrome P450IIC4 cDNA and induction by phenobarbital of related hepatic mRNA levels.

We have cloned cDNA containing a partial sequence of a rabbit cytochrome P-450 (designated cytochrome P450IIC4) cDNA that is a member of the cytochrome P450IIC subfamily. The cDNA contains 770 bp of which the first 429 code for the C-terminal 143 amino acids of cytochrome P450IIC4. The protein coding region of the cDNA is 98% homologous with that of cytochrome P450IIC5 and the 3' untranslated region is about 90% homologous. In contrast to the constitutive isozyme, cytochrome P450IIC5, mRNA in the liver that hybridized to the 3' untranslated region of cytochrome P450IIC4 cDNA was increased about 8-fold 24 hours after a single injection of phenobarbital.

Animals↗
PubMed1987

Mutations in the NH2-terminal domain of the signal peptide of preproparathyroid hormone inhibit translocation without affecting interaction with signal recognition particle.

The amino-terminal domain of a eukaryotic signal peptide, from bovine parathyroid hormone, was altered by in vitro mutagenesis of the cDNA. The function of "internalized" signal sequence mutants and of deletion mutants was assayed using an in vitro translation-translocation system. The addition of 11 amino acids to the NH2 terminus of the signal peptide did not prevent normal processing of the precursor protein, whereas a 23-amino acid extension blocked processing. These data suggest that the NH2-terminal sequences of internal signal peptides must be permissive of the signal function. Deletion of 6 NH2-terminal amino acids from the signal peptide had no effect on its cleavage by microsomal membranes, but removal of 10 or 13 amino acids, including all charged residues prior to the hydrophobic core, prevented processing. For both the extension and deletion mutations, processed proteins were protected from proteolytic digestion, whereas unprocessed forms were not, which indicated that the unprocessed mutant proteins were not translocated across the microsomal membrane. Translation of both the extension and deletion translocation-deficient mutants was arrested by signal recognition particle, and salt-washed microsomal membranes reversed the translational arrest. These data demonstrate that the NH2-terminal domain is not required for the interaction of signal recognition particle with the signal peptide or with signal recognition particle receptor, but is required for formation of a maximally translocation-competent complex with the microsomal membrane.

Amino Acid Sequence↗
PubMed1987

The P450 gene superfamily: recommended nomenclature.

A nomenclature for the P450 gene superfamily is proposed based on evolution. Recommendations include Roman numerals for distinct gene families, capital letters for subfamilies, and Arabic numerals for individual genes. An updating of this list, which presently includes 65 entries, will be required every 1-2 years. Assignment of orthologous genes is presently uncertain in some cases--between widely diverged species and especially in the P450II family due to the large number of genes. As more is known, it might become necessary to change some gene assignments that are based on our present knowledge.

Alleles↗
PubMed1987

Protein-binding sites within the 5' DNase I-hypersensitive region of the chicken alpha D-globin gene.

We mapped at high resolution and as a function of development the hypersensitive domain in the 5'-flanking region of the chicken alpha D-globin gene and determined the specific protein-binding sites within the domain. The domain extends from -130 to +80 nucleotides (nt) relative to the cap site. DNase I footprinting within intact embryonic erythrocyte nuclei revealed a strongly protected area from -71 to -52 nt. The same area was weakly protected in adult nuclei. A factor was present in extracts of erythrocyte nuclei from both embryos and adults that protected the sequence AAGATAAGG (-63 to -55 nt) in DNase I footprinting experiments; at higher concentrations of extract, sequences immediately adjacent (-73 to -64 and -53 to -38) were also protected. The same pattern of binding was revealed by gel mobility shift assays. The identical AAGATAAGG sequence is found in the 5'-flanking region of the beta rho gene; it competed for binding of the alpha D-specific factor, suggesting that regulatory elements are shared.

Animals↗
PubMed1986

Evidence that rabbit cytochrome P-450 K is encoded by the plasmid pP-450 PBc2.

The complete coding sequence for P-450 PBc2 was inserted into a T7-phage promoter system, and a capped cRNA was generated using T7 RNA polymerase. The P-450 PBc2 cRNA was translated in a rabbit reticulocyte lysate. The in vitro translation product was indirectly immunoprecipitated by the monoclonal antibodies 2F5 and 3C3 that recognize P-450 K. SDS-polyacrylamide gel electrophoresis revealed that the translated protein product exhibits the same relative electrophoretic mobility as P-450 K. The N-terminal amino acid sequence was determined to be MDLVVVLGL-LS-LLLLSL- for P-450 K immunopurified from rabbit kidney using the monoclonal antibody 2F5. This sequence agrees with the predicted amino acid sequence derived from the P-450 PBc2 cDNA. These results indicate that P-450 K or a protein closely related to P-450 K is encoded by the plasmid pP-450 PBc2.

Amino Acid Sequence↗
PubMed1986

The selection of the first AUG as the initiator of eucaryotic mRNAs translation is favored by a 5'-terminal cap group and a purine in the -3' position.

We have constructed a series of plasmids containing variations in the DNA that codes for the translation initiation region of preproparathyroid hormone. Gel electrophoretic analyses of the character and extent of synthesis of various modified preproparathyroid hormone like proteins derived from the mRNAs transcribed from these plasmids reveal that the presence of a 5'-terminal cap group on mRNA's facilitates recognition of the most 5'-terminal AUG sequence on a mRNA and that AUG sequences within the consensus sequence PuXXAUGPu are favored sites for the initiation of mRNA translation.

Base Sequence↗
PubMed1986

Endonuclease VII resolves Y-junctions in branched DNA in vitro.

Endonuclease VII (gp 49 of phage T4) resolves four-way junctions in branched DNAs. We have extended our investigations of the specificity of endo VII and tested its activity with three-way junctions (Y-structures) constructed in vitro. Both 'closed' and 'open' Y-structures were made, absolutely identical in sequence but differing from each other by a single nick in one of the three arms. Pure Y-structures were obtained on a preparative scale by annealing plus and minus strands from two M13mp strains. One strain has an inverted repeat of 2 X 31 nucleotides cloned into the single EcoRI site while in the other strain this repeat is absent. The structures were used in reactions with endo VII, which recognizes the branch point of both structures and introduces a characteristic number of nicks, 3' to the junction in each arm of the structure. Strong and weak sites could be distinguished and the cleavage pattern differed significantly between the two structures. The observed resolution of Y-junctions by endo VII in vitro is compatible with a model for the resolution of recombinant Y-branches in DNA.

Base Sequence↗
PubMed1986

Structure of genes in the cytochrome P-450PBc subfamily: conservation of intron locations in the phenobarbital-inducible family.

The structures of P-450 genes in the rabbit phenobarbital-inducible subfamily of P-450 genes, defined by the cytochrome P-450PBc cDNAs, have been determined by restriction mapping and nucleotide sequencing. A genomic clone corresponding to the 5'-flanking region and the first six exons of the cytochrome P-450PBc2 gene has been examined in detail, and two other clones corresponding to exons 7-9 and the 3'-flanking region of the cytochrome P-450PBc3 and cytochrome P-450(form 1) genes have been characterized less extensively. The intron sequences adjacent to the exons are typical of vertebrate nuclear genes and contain GT and AG at their 5' and 3' ends, respectively. The intron locations in these genes are identical to those in the genes for rat cytochrome P-450b/e, members of the other phenobarbital-inducible subfamily, but completely different from those in the 3-methylcholanthrene-inducible cytochrome P-450 genes. Repetitive sequences are present in at least three introns of the cytochrome P-450PBc2 gene, but not in exons or the 5'-flanking region. The site of transcription initiation for the cytochrome P-450PBc2 gene was determined by primed reverse transcription and S1 mapping. Although the mRNA appeared to be heterogenous at the 5' terminus, the most probable start site is 18 bp downstream from a canonical TATA sequence and 23 bp upstream from the initiator ATG codon. The nucleotide sequence of 800 bp of the 5'-flanking region of the cytochrome P-450PBc2 gene has been determined. This region shares little homology with the 5'-flanking region of rat cytochrome P-450b/e genes, which are 55-60% homologous in the protein-coding region with the members of the P-450PBc subfamily. The amino-terminal sequence of cytochrome P-450PBc2, which could not be determined from the cDNA clones, was derived from the gene sequence. Comparison of the amino-terminal amino acid sequence of P-450PBc2 with those of other mammalian P-450s indicates that the rat isozymes f, g, h, i, and PB1 are in the same subfamily as the rabbit isozymes form 1, form 3b or P-450PBc3, P-450PBc1, and P-450PBc2.

Amino Acid Sequence↗
PubMed1986

Molecular biology of parathyroid hormone.

The entire biosynthetic pathway of PTH has been elucidated from the determination of the chromosomal location to the eventual secretion of the hormone from the cell. The human gene is present on the short arm of chromosome 11, and restriction site polymorphisms near the gene have been detected. The PTH genes and cDNAs have been isolated and characterized in the bovine, human, and rat species. The gene contains two introns, which are in the same position in each species, and dissect the gene into 3 exons that code, respectively, for the 5' untranslated region, the signal peptide, and PTH plus the 3' untranslated region. The mRNAs are about twice as long as necessary to code for preProPTH and contain a 7-methylquanosine cap at the 5' terminus and polyadenylic acid at the 3' terminus. The 5' termini of the bovine and human mRNAs are heterogeneous at the 5' terminus, the basis of which is two TATA sequences in the 5' flanking regions of the gene. In contrast, the rat gene contains a single TATA sequence and the mRNA has a single 5' terminus. The initial translational product of the mRNA is preProPTH, and the pre-peptide of 25 amino acids is equivalent to signal peptides of other secreted and membrane proteins. The genes of the three species are very homologous in the region that codes for preProPTH. Substantial homology is also retained in the gene flanking regions, introns, and mRNA untranslated regions. Silent sites are also conserved more than would be expected, particularly between the human and bovine sequences. The bovine and human sequences are more closely related than the rat is to either the human or bovine. These studies of the basic molecular biology of PTH will provide the framework for future analysis of significant biological and medical questions. In vitro mutagenesis techniques should soon provide information about the elements of the gene involved in regulating transcription and about functional elements of the signal peptide. Eventually, signals involved in directing the ProPTH molecule to secretory granules as well as the biologically active regions of PTH, itself, will be examined by these methods. The molecular biological studies, combined with the development of dispersed cell cultures, provide the opportunity to study the effects of chronic changes in calcium on gene transcription and mRNA metabolism. The restriction site polymorphisms associated with the human PTH gene will allow a search for correlations between PTH gene structure and parathyroid disease.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1985

Single stranded DNA SP6 promoter plasmids for engineering mutant RNAs and proteins: synthesis of a 'stretched' preproparathyroid hormone.

The intergenic region of bacteriophage f1 has been subcloned into the bacteriophage SP6 promoter plasmids, pSP64 and pSP65, in both orientations. Coinfection of E. coli with these SP6 promoter/phage f1 chimeric plasmids and the interference resistance phage, IR1, results in the replication and secretion of the pSP6.f1 plasmids as single stranded DNA. Bovine preProPTH cDNAs in both the native form and a form containing an insertion of 117 base pairs in the protein coding region have been inserted in these plasmids. The RNA transcribed from the SP6.f1/preProPTH cDNA constructs was efficiently translated in the wheat germ or reticulocyte cell free systems without addition of a 7-methylguanosine cap to the RNA. In the presence of dog pancreatic or chicken oviduct microsomal membranes, conversion of the resultant pre-proteins to pro-proteins was observed. Confirmation of the "mutated" preProPTH cDNA was determined by dideoxyribonucleotide DNA sequencing of single stranded plasmid DNA. These vectors are suitable for the efficient biosynthesis of large amounts of single or double stranded DNA, and translationally active RNA. The combined properties of single stranded DNA replication and the SP6 promoter simplify the engineering of mutant RNAs and their corresponding proteins. In addition, single stranded DNA or RNA corresponding to either complementary strand may be synthesized as nucleic acid hybridization probes.

Animals↗
PubMed1984

Differential induction and tissue-specific expression of closely related members of the phenobarbital-inducible rabbit cytochrome P-450 gene family.

We have examined the tissue-specific expression of three rabbit genes that are closely related members of a subfamily of the phenobarbital-inducible cytochrome P-450 gene family. Analysis of the levels of mRNA in liver revealed that (a) cytochrome P-450PBc1 mRNA was not detectable in livers from control animals but was present in livers from animals treated with phenobarbital, (b) cytochrome P-450PBc2 was present in control tissue and was increased by about 3-fold 24 h after phenobarbital treatment, and (c) the levels of cytochrome P-450PBc3 mRNA was the same in livers from control and treated animals. In the kidney, only P-450PBc2 mRNA was detected at a level 15% of that in the liver, and the levels increased about 3-fold after phenobarbital treatment. None of the mRNAs was detected in lung tissue. Multiple species of RNA were observed that hybridized to probes for cytochrome P-450PBc1 and P-450PBc2 cDNAs by Northern blot analysis ranging in size from 2300 to 4000 nucleotides. Differential sites for polyadenylation probably cause the heterogeneity in size. A single species of RNA of 2200 nucleotides that hybridized to cytochrome P-450PBc3 cDNA probes was observed. These data demonstrate that three closely related cytochrome P-450 genes are differentially responsive to phenobarbital treatment and that they exhibit different tissue-specific patterns of expression.

Animals↗