Biomedical subjects

B Kemper

Publications and source records attributed to B Kemper.

At least 91 records · Page 5Linked to original sources

PubMed1992

Transcriptional regulatory elements for basal expression of cytochrome P450IIC genes.

To analyze the transcriptional regulatory elements in rabbit cytochrome P450IIC genes, varying lengths of the 5'-flanking regions of CYP2C1 and CYP2C2 were fused to a luciferase reporter gene. Promoter activity was assayed by transfection into HepG2 cells, a hepatic cell line, and monkey kidney COS-1 cells, a nonhepatic cell line. Activity of the CYP2C1 promoter in HepG2 cells increased slightly with progressive 5' deletions of the 5'-flanking region from nucleotide -3600 to -1500 relative to the transcription start site. Additional deletions to -900, -358, and -116 each reduced activity by about 50%, and deletion of the sequence from -116 to -67 reduced activity by a factor of 12. Activity of the CYP2C2 promoter increased about 3-fold with progressive 5' deletions of sequence from nucleotide -3500 to -410. In contrast, deletions of sequences from -251 to -193 and from -133 to -64 reduced promoter activity by factors of 2 and 8, respectively. In COS-1 cells, the maximum activities of the CYP2C1 and CYP2C2 promoters normalized to a Rous sarcoma viral promoter were about 10-20% of that in the HepG2 cells. The changes in activity between different constructions in COS-1 cells largely paralleled those in the HepG2 cells except for deletions of the sequences -133 to -64 and -116 to -67 for CYP2C1 and CYP2C2, respectively, which produced the largest reduction of promoter activity in HepG2 cells but had little effect in COS-1 cells. These results show that HepG2-specific regulatory elements are present in the regions between -120 and -65 in both genes. Nuclear proteins from HepG2 cells, but not from COS-1 cells, bound to sequences within these regions, and the binding was inhibited by an oligonucleotide containing a sequence conserved in rabbit P450IIC genes which has been designated the HepG2-specific P450 2C factor-1 (HPF1) motif. Mutation of this sequence eliminated the binding of nuclear proteins and reduced transcriptional activity 25-fold. The HPF1 binding sequence is conserved in CYP2A, CYP2C, and CYP2D genes and resembles the binding motif for hepatic nuclear factor-4. These results demonstrate that CYP2C1 and CYP2C2 contain several potential regulatory elements for basal expression, including one HepG2-specific sequence that may be important for liver expression.

Animals↗

PubMed1992

T4 endonuclease VII resolves cruciform DNA with nick and counter-nick and its activity is directed by local nucleotide sequence.

Endonuclease VII of phage T4 resolves Holliday structures in vitro by nicking pairs of strands across the junction. We report here analyses of this reaction between endonuclease VII and a Holliday structure analogue, made in vitro from synthetic oligonucleotides. The enzyme cleaves the structure in a non-concerted way and nicks each strand independently. Combinations of nicks with counter-nicks in strands across the junction resolve the construct. The specificity of the enzyme for DNA secondary structures was tested with a series of branched molecules made from oligonucleotides with the same nucleotide sequence in one strand. Results show that the number, location and relative cleavage efficiencies depend largely on the local nucleotide sequence, rather than on the branch type. In particular, endonuclease VII cleaves a complete four-armed cruciform as efficiently as a three-armed Y-junction or its derivatives, a semi-Y, a fork with two single-strand overhangs, a single-strand overhang, and a nicked DNA. However, exchange or addition of one or more nucleotides within the cleavage area flanking the structural signal for endonuclease VII strongly affects the cleavage pattern as well as their relative efficiency of usage. Examples with a single-stranded overhang are presented and show in summary that the enzyme has a fivefold preference for pyrimidines rather than purines.

Base Sequence↗

PubMed1992

Identification and characterization of yeast mutants and the gene for a cruciform cutting endonuclease.

An assay was developed that detected DNA cruciform cutting endonuclease activity in crude extracts of Saccharomyces cerevisiae. A collection of temperature-sensitive strains was screened using this assay, and a mutant lacking the activity was found. The mutation leading to the enzymatic defect was mapped to the left arm of chromosome XI within 3 cM of the centromere. Cloning of the gene for this endonuclease was achieved by chromosome walking from the nearby PUT3 locus. The gene, called CCE1 (cruciform cutting endonuclease), was sequenced and found to have an open reading frame encoding a 41 kDa protein. The amino acid sequence of this eukaryotic endonuclease shows homology neither to its prokaryotic counterparts nor to other proteins in available databases. A cce1 null mutant has no obvious growth defect, and despite the ability of the CCE1 enzyme to cleave Holliday junction analogs, the mutant shows no defect in meiotic or mitotic recombination. A second cruciform cutting activity was detected in extracts from a cce1 null mutant, indicating that yeast has at least two such enzymes. The only phenotype observed for cce1 mutants is a higher than normal frequency of appearance of petite cells, suggesting that the CCE1 protein is important for the maintenance of mitochondrial DNA.

Amino Acid Sequence↗

PubMed1992

Frontal lobe function in amyotrophic lateral sclerosis: a neuropsychologic and positron emission tomography study.

In this study the regional cerebral glucose utilization and the neuropsychological performance of patients with amyotrophic lateral sclerosis (ALS) was investigated. Special attention was given to neuropsychological tests thought to mirror frontal lobe dysfunction. The regional cerebral glucose utilization was studied in 18 patients using high-resolution positron emission tomography. Clinically all patients displayed upper and lower motor neurone signs. In ALS patients glucose metabolism was significantly reduced in the frontal and in the entire cortex compared with controls; no changes were seen in the cerebellum. Comprehensive neuropsychological assessment of ALS patients compared to a pair matched control group revealed mild frontal dysfunction which in part significantly correlated with reduced glucose metabolism in the cortex and subcortical structures. We conclude that in patients with ALS, glucose consumption is decreased in parts of the brain other than the motor cortex accompanied by mild neuropsychological deficits based on the tests employed in this study.

Adult↗

PubMed1991

Differences in deoxyribonuclease I hypersensitive sites in phenobarbital-inducible and constitutive rabbit P450IIC genes.

DNase I hypersensitivity of nuclear chromatin near the rabbit cytochrome P450IIC genes was investigated by indirect end-labeling genomic Southern analysis. Major DNase I hypersensitive sites were observed in proximal (-200 base pairs) and distal (-2000 to -2200 base pairs) regions of the genes. The presence of the proximal site correlated well with the expression states of the individual genes. In contrast, the distal site was present in DNA from both liver and kidney nuclei and in untreated and phenobarbital-treated animals irrespective of the expression state of the genes. However, the distal site was present only in genes that respond to phenobarbital (cytochromes P450IIC1 and P450IIC2) and was not detected in the constitutive cytochrome P450IIC3 gene. The nucleotide sequences of 500 base pairs in the distal site regions of cytochromes P450IIC1 and P450IIC2 were 67% similar, and hepatocyte nuclear factor 1 like motifs were conserved in these two sequences. These results are consistent with the hypothesis that distal regions cooperate with the proximal promoter regions in the regulation of cytochrome P450IIC gene expression.

Animals↗

PubMed1991

Model for the interaction of DNA junctions and resolving enzymes.

Four-way DNA junctions are thought to be important intermediates in a number of recombination processes. Resolution of these junctions occurs by cleavage of two strands of DNA to generate two duplex molecules. The interaction between DNA junctions and resolving enzymes appears to be largely structure-specific, reflecting a molecular recognition on a significant scale. We propose a working model for this interaction that takes account of the present state of knowledge of the structure of the DNA junction, and the substrate requirements of the enzymes. We note that three different enzymes introduce cleavages at phosphodiester bonds that are presented on one side of the molecule, suggesting that the enzymes selectively interact with this face of the junction. By forcing a junction of constant sequence to adopt one or other of the two possible antiparallel isomers, we show that the junction is cleaved in such a way as to suggest a constant mode of interaction with the protein that is dependent on structure rather than sequence. We propose that the feature that is recognized is a mutual inclination of two DNA helices at approximately 120 degrees. We show that a number of DNA substrates that contain similar inclined helices, such as a three-way junction, bulged duplexes and a duplex that is curved because of repeated runs of oligoadenine sequences, are each cleaved by phage T4 endonuclease VII. This mode of DNA-protein interaction could be significant in either recombination or DNA repair processes.

Animals↗

PubMed1991

A hypervariable region of P450IIC5 confers progesterone 21-hydroxylase activity to P450IIC1.

Cytochrome P450IIC5 is a hepatic progesterone 21-hydroxylase while the 95% identical P450IIC4 has a greater than 10-fold higher Km for progesterone 21-hydroxylation and the 74% identical P450IIC1 does not hydroxylate progesterone at detectable rates. Previous work demonstrated that the apparent Km of P450IIC4 for progesterone 21-hydroxylation can be markedly improved by replacing a valine at position 113 with an alanine which is present at this position in P450IIC5. In the present studies, a single point mutation in cytochrome P450IIC1 that changed valine at position 113 to alanine conferred progesterone 21-hydroxylase activity to this enzyme. Although the catalytic activity was less than that of P450IIC5, these results indicate the residue 113 plays a critical role in the determination of the substrate/product selectivity in subfamily IIC P450s. By alignment with the sequence of P450cam, the segment of the polypeptide, residues 95-123, containing residue 113 corresponds to a substrate-contacting loop in the bacterial enzyme. The region containing residue 113, which is highly variable among family II P450s, may also be a substrate-contacting loop in the mammalian cytochromes P450. The exchange of this hypervariable region of cytochrome P450IIC1, residues 95-123, with that of P450IIC5 enhanced the 21-hydroxylase activity of the cells transfected with this chimera to levels similar to those of cells transfected with the plasmid encoding P450IIC5. Kinetic analysis of microsomes isolated from the transfected cells showed that the apparent Km for progesterone 21-hydroxylation of the chimera was indistinguishable from that of P450IIC5.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1991

Assembly and characterization of five-arm and six-arm DNA branched junctions.

DNA branched junctions have been constructed that contain either five arms or six arms surrounding a branch point. These junctions are not as stable as junctions containing three or four arms; unlike the smaller junctions, they cannot be shown to migrate as a single band on native gels when each of their arms contains eight nucleotide pairs. However, they can be stabilized if their arms contain 16 nucleotide pairs. Ferguson analysis of these junctions in combination with three-arm and four-arm junctions indicates a linear increase in friction constant as the number of arms increases, with the four-arm junction migrating anomalously. The five-arm junction does not appear to have any unusual stacking structure, and all strands show similar responses to hydroxyl radical autofootprinting analysis. By contrast, one strand of the six-arm junction shows virtually no protection from hydroxyl radicals, suggesting that it is the helical strand of a preferred stacking domain. Both junctions are susceptible to digestion by T4 endonuclease VII, which resolves Holliday junctions. However, the putative helical strand of the six-arm junction shows markedly reduced cleavage, supporting the notion that its structure is largely found in a helical conformation. Branched DNA molecules can be assembled into structures whose helix axes form multiply connected objects and networks. The ability to construct five-arm and six-arm junctions vastly increases the number of structures and networks that can be built from branched DNA components. Icosahedral deltahedra and 11 networks with 432 symmetry, constructed from Platonic and Archimedean solids, are among the structures whose construction is feasible, now that these junctions can be made.

Base Sequence↗

PubMed1991

Cell-free analysis of targeting of cytochrome P450 to microsomal membranes.

Analysis of P450 biosynthesis in cell-free systems has been useful in studying the mechanism of the targeting of P450 to the membrane. These techniques have allowed the demonstration that the N-terminal region of P450 is an uncleaved signal sequence for SRP-dependent membrane insertion. Lack of protection from protease indicated that most of the P450 is on the cytoplasmic side of the membrane. Substitution of basic amino acids at the P450 N terminus or replacing the N terminus of P450 with a secretory signal sequence results in partial or complete translocation across the membrane. This result indicates that at most one and probably none of the internal hydrophobic regions function as stop-transfer signals, which would be present if the protein spanned the membrane more than once.

Animals↗

PubMed1990

Characterization of a bimobile DNA junction.

We present here a chemical and enzymatic footprinting analysis of a branched DNA molecule formed from four complementary 50-mer strands. These strands are designed to form a stable junction, in which two steps of branch point migration freedom are possible. Exposure of the junction to Fe(II).EDTA shows protection of 3 or 4 residues in each strand at the branch, while two resolvase enzymes (endonuclease VII from phage T4 and endonuclease I from phage T7), cleave all four strand near the branch. Chemical footprinting of this junction using the reagents MPE.Fe(II) and (OP)2Cu(I) shows that the branch site is hyper-reactive to cutting induced by these probes as it is in an immobile four-arm junction. The effects involve more residues than in the immobile case. In the absence of divalent cations, the structure of the junction alters, sites of enhanced cleavage by MPE.Fe(II) and (OP)2Cu(I) disappear, and purines at the branch become reactive to diethyl pyrocarbonate. Our interpretation of these results is based on the properties of immobile junction analogs and their response to these probes. In the presence of Mg2+, the three migrational isomers coexist, each probably in the form of a 2-fold symmetric structure with two helical arms stacked.

Base Sequence↗

PubMed1990

Resolution of Holliday junction analogs by T4 endonuclease VII can be directed by substrate structure.

Endonuclease VII is an enzyme from bacteriophage T4 capable of resolving four-arm Holliday junction intermediates in recombination. Since natural Holliday junctions have homologous (2-fold) sequence symmetry, they can branch migrate, creating a population of substrates that have the branch point at different sites. We have explored the substrate requirements of endonuclease VII by using immobile analogs of Holliday junctions that lack this homology, thereby situating the branch point at a fixed site in the molecule. We have found that immobile junctions whose double-helical arms contain fewer than nine nucleotide pairs do not serve as substrates for resolution by endonuclease VII. Scission of substrates with 2-fold symmetrically elongated arms produces resolution products that are a function of the particular arms that are lengthened. We have confirmed that the scission products are those of resolution, rather than nicking of individual strands, by using shamrock junction molecules formed from a single oligonucleotide strand. A combination of end-labeled and internally labeled shamrock molecules has been used to demonstrate that all of the scission is due to coordinated cleavage of DNA on opposite sides of the junction, 3' to the branch point. Endonuclease VII is known to cleave the crossover strands of Holliday junctions in this fashion. The relationship of the long arms to the cleavage direction suggests that the portion of the enzyme which requires the minimum arm length interacts with the pair of arms containing the 3' portion of the crossover strands on the bound surface of the antiparallel junction.

Base Sequence↗

PubMed1990

Interaction of a four-way junction in DNA with T4 endonuclease VII.

The binding of a synthetic four-way junction in DNA by T4 endonuclease VII has been studied using gel retardation and footprint analysis. Two specific protein-DNA complexes have been observed, but only one is stable in the presence of moderate concentrations of salt. The footprint of T4 endonuclease VII in the salt-resistant complex has been probed using hydroxyl radicals generated by the reaction of iron(II)/EDTA with hydrogen peroxide. The hydroxyl radical cleavage pattern indicates protection of approximately 5 residues in two strands that are diametrically opposed across the junction point.

Base Sequence↗

PubMed1990

Cleavage specificity of bacteriophage T4 endonuclease VII and bacteriophage T7 endonuclease I on synthetic branch migratable Holliday junctions.

Holliday junctions are intermediate structures that are formed and resolved during the process of genetic recombination. To investigate the interaction of junction-resolving nucleases with synthetic Holliday junctions that contain homologous arm sequences, we constructed substrates in which the junction point was free to branch migrate through 26 base-pairs of homology. In the absence of divalent cations, we found that both phage T4 endonuclease VII and phage T7 endonuclease I bound the synthetic junctions to form specific protein-DNA complexes. Such complexes were not observed in the presence of Mg2+, since the Holliday junctions were resolved by the introduction of symmetrical cuts in strands of like polarity. The major sites of cleavage were identified and found to occur within the boundaries of homology. T4 endonuclease VII showed a cleavage preference for the 3' side of thymine bases, whereas T7 endonuclease I preferentially cut the DNA between two pyrimidine residues. However, cleavage was not observed at all the available sites, indicating that in addition to their structural requirements, the endonucleases show strong site preferences.

Base Sequence↗

PubMed1990

Structure of the rabbit cytochrome P450IIC3 gene, a constitutive member of the P450IIC subfamily.

Fragments of rabbit DNA have been cloned which encompass the gene for rabbit cytochrome P450IIC3. Cytochrome P450IIC3 is a constitutive member of the cytochrome P450IIC subfamily which contains both constitutive and phenobarbital-responsive genes. The cytochrome P450IIC3 gene spans at least 25 kbp and contains 8 introns which have 5'GT's and 3'AG's and are located in the same positions as in other family II genes. The 5' flanking region contains a consensus TATA site about 25 bp from the RNA initiation site which was mapped by the primer extension method. Other potential regulatory sequences include a CCAAT sequence and sequences similar to binding sites for the liver-specific factor HNF-1 and the general transcription factors AP-1 and OCT. The gene sequence differs from the cytochrome P450IIC3 cloned cDNA sequence in only 2 of 1400 nucleotides, 1 of which results in an amino acid change. Since sequences coding for exon 1 and part of exon 2 were not present in the cloned cytochrome P450IIC3 cDNA, a 475-nucleotide fragment of the 5' end of the cDNA was amplified by the polymerase chain reaction, cloned, and sequenced. The sequence of this cDNA was identical with the gene exon sequence. The protein sequence derived from the gene differs in nine positions from that determined directly for the protein. A comparison of the amino acid sequence by exons with other cytochrome P450IIC proteins revealed that, in general, the similarity was greater in C-terminal exons than in N-terminal ones.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1990

Enzymatic formation and resolution of Holliday junctions in vitro.

E. coli RecA protein promotes homologous pairing and reciprocal strand exchange reactions between duplex DNA molecules in vitro. Reaction intermediates contain Holliday junctions that are driven along the DNA at a maximal rate approaching 1000 bases per minute. T4 endonuclease VII cleaves Holliday junctions in vitro, and its inclusion in RecA-mediated reactions leads to the rapid formation of heteroduplex products. Product analysis indicates patch and splice recombinant molecules similar to those expected from in vivo recombination events. The combined formation and resolution of Holliday junctions has led us to propose a model for resolution based on the structure of RecA-DNA helices. One feature of this model is that resolution, which gives rise to the two types of recombinant product, may occur without need for isomerization of the junction.

Bacteriophage phi X 174↗

PubMed1989

Cruciform cutting endonucleases from Saccharomyces cerevisiae and phage T4 show conserved reactions with branched DNAs.

We have purified a cruciform DNA resolving endonuclease (Endo X3) greater than 1000-fold from crude extracts of mitotically growing Saccharomyces cerevisiae. The enzyme shows high specificity for DNAs with secondary structures and introduces characteristic patterns of staggered 'nicks' in the immediate vicinity of the structure. The following substrates were analyzed in detail: (i) naturally occurring four-way X junctions in cruciform DNA of a supercoiled plasmid; (ii) synthetic four-way X junctions with arms of 9 bp; (iii) synthetic three-way Y junctions with arms of 10 bp; and (iv) heteroduplex loops with 19 nucleotides in the loop. Cleavages were always found in the double stranded portion of the DNA, located immediately adjacent to the junction of the respective structure. The Endo X3 induced cleavage patterns are identical or very similar to the cleavage patterns induced in the same substrates by endonuclease VII (Endo VII) from phage T4. Furthermore, the activity of Endo X3 is completely inhibited in the presence of anti-Endo VII antiserum. Endo X3 has an apparent mol. wt of 43,000 daltons, determined by gel filtration and of approximately 18,000 daltons in SDS--polyacrylamide gels. Maximum activity of the enzyme was obtained in the presence of 10 mM MgCl2 at 31 degrees C in Tris-HCl buffer over a broad pH range with a maximum approximately 8.0. About 70% of maximal activity was obtained when Mg2+ was replaced by equimolar amounts of Mn2+ or Ca2+.

Base Sequence↗

PubMed1989

Parallel effects of signal peptide hydrophobic core modifications on co-translational translocation and post-translational cleavage by purified signal peptidase.

The length of the hydrophobic core of the bovine parathyroid hormone signal peptide was modified by in vitro mutagenesis. Extension of the hydrophobic core by three amino acids at the NH2-terminal end had little effect on the proteolytic processing of the signal peptide by microsomal membranes. Deletion of 6 of the 12 amino acids in the core eliminated translocation and processing of the modified protein. Deletion of pairs of amino acids across the core resulted in position-dependent inhibition of signal activity unrelated to hydrophobicity but inversely related to the hydrophobic moments of the modified cores. Deletions in the NH2-terminal region of the core were strongly inhibitory for proteolytic processing whereas deletions in the COOH-terminal region had no effect or increased processing when assessed either co-translationally with microsomal membranes or post-translationally with purified hen oviduct signal peptidase. Deletion of cysteine 18 and alanine 19 increased processing, but deletion of cysteine alone or substitution of leucine for cysteine did not increase processing more than deletion of both residues at 18 and 19. Translations of the translocation-defective mutants with pairs of amino acids deleted in a wheat germ system were inhibited by addition of exogenous signal recognition particle suggesting that interactions of the modified signal peptides with signal recognition particle were normal. The position-dependent effects of the hydrophobic core modifications indicate that structural properties of the core in addition to hydrophobicity are important for signal activity. The parallel effects of the modifications on co-translational translocation and post-translational processing by purified signal peptidase suggest that proteins in the signal peptidase complex might be part of, or intimately associated with, membrane proteins involved in the translocation. A model is proposed in which the NH2-terminal region of the hydrophobic core binds to one subunit of the signal peptidase while the other subunit catalyzes the cleavage.

Amino Acid Sequence↗

PubMed1989

NH2-terminal substitutions of basic amino acids induce translocation across the microsomal membrane and glycosylation of rabbit cytochrome P450IIC2.

Insertion of rabbit cytochrome P450IIC2 and its modified form, [2-lys,3-arg]P450IIC2, into microsomal membranes was studied in an in vitro transcription/translation/translocation system. Cytochrome P450IIC2, synthesized in the presence of chicken oviduct microsomal membranes, was resistant to extraction by alkaline solutions, but was sensitive to proteolytic digestion. In contrast, when [2-lys,3-arg]-P450IIC2 was synthesized in the presence of membranes, two new species migrating more slowly during gel electrophoresis were observed. After treatment with endoglycosidase H, the more slowly migrating species comigrated with [2-lys,3-arg]P450IIC2 synthesized in the absence of membranes, indicating that the proteins had been glycosylated. Both the glycosylated and nonglycosylated forms of [2-lys,3-arg]P450IIC2 were resistant to proteolytic digestion and to extraction from the membranes by alkaline solutions. Similar results were obtained for a truncated species, [2-lys,3-arg]P450IIC2(1-55), except that only a single glycosylated species was observed, consistent with the single remaining glycosylation site. In contrast to the proteolytic processing observed previously in a hybrid [2-lys,3-arg]P450IIC2/parathyroid hormone protein, little or no cleavage of the NH2-terminal peptide of [2-lys,3-arg]P450IIC2 was observed in the presence of membranes. Since cleavage in the hybrid protein occurred after glycine 25, which is derived from [2-lys,3-arg]P450IIC2, cytochrome P450 sequences COOH terminal to the cleavage site must decrease cleavage efficiency. These results demonstrate that cytochrome P450, which is normally localized on the cytoplasmic side of the membrane, can be entirely translocated to the luminal side when two basic amino acids precede the hydrophobic core of its NH2-terminal insertion/stop-transfer signal. None of the several internal hydrophobic regions of cytochrome P450, previously proposed as membrane spanning, function as a stop-transfer signal.

Amino Acid Sequence↗