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Biomedical subjects

G Zubay

Publications and source records attributed to G Zubay.

At least 19 recordsLinked to original sources
PubMed1999

Nucleoside phosphorylation: a feasible step in the prebiotic pathway to RNA.

Plausible prebiotic conditions for the phosphorylation of nucleosides by inorganic phosphate were reported by Lohrmann and Orgel in 1971. This reaction was carried out on heated dry films and promoted by urea. The major products formed were nucleoside-2:3 cyclicPs;5-NMPs and other derivatives were also formed. Minor modifications of the Lohrmann and Orgel system have resulted in the preferential formation of 5-NMPs. In this modified system a 2-fold preference for phosphorylation of the 5-OH group over the 3(2)-OH group was observed and the formation of other derivatives was minimized. The small amounts of bis compounds that were formed in this system could be quantitatively removed by selective binding to the mineral hydroxylapatite at moderate ionic strengths. It was also discovered that under hydrolytic conditions there was a 3:1 preference for removal of phosphates attached to the 3-OH group over the 5-OH group. A recycling procedure for obtaining additonal 5-NMPs from bis compounds and 3-NMPs is proposed.

Adenosine↗
PubMed1998

Studies on the lead-catalyzed synthesis of aldopentoses.

The object of this work was to find an efficient means of synthesizing ribose in a manner that could be considered prebiotic. The starting point for synthesis was an aqueous solution of formaldehyde. Heretofore the most frequently used catalyst for this purpose has been calcium hydroxide. Unfortunately this system produces a wide array of products in addition to ribose which constitutes 1% or less of the final product. Attempts were made to find more mild conditions under which the formaldehyde could be reacted. Magnesium hydroxide suspensions were used for this purpose. Formaldehyde does not yield any sugars when incubated in magnesium hydroxide suspensions alone. However, if the magnesium hydroxide suspension was supplemented with doubly charged lead salts and catalytic amounts of any intermediate in the prebiotic pentose pathway, aldopentoses accounted for 30 per cent or more of the final product. The presence of lead in the incubation mixture also accelerated a number of other reactions including the interconversion of the four common aldopentoses, ribose, arabinose, lyxose and xylose.

Acetophenones↗
PubMed1995

Consecutive low-usage leucine codons block translation only when near the 5' end of a message in Escherichia coli.

Insertion of nine consecutive low-usage CUA leucine codons after codon 13 of a 313-codon test mRNA strongly inhibited its translation without apparent effect on translation of other mRNAs containing CUA codons. In contrast, nine consecutive high-usage CUG leucine codons at the same position had no apparent effect, and neither low- nor high-usage codons affected translation when inserted after codon 223 or 307. Additional experiments indicated that the strong positional effect of the low-usage codons could not be accounted for by differences in stability of the mRNAs or in stringency of selection of the correct tRNA. The positional effect could be explained if translation complexes are less stable near the beginning of a message: slow translation through low-usage codons early in the message may allow most translation complexes to dissociate before they read through.

Blotting, Northern↗
PubMed1995

Binding of adenine and adenine-related compounds to the clay montmorillonite and the mineral hydroxylapatite.

The first living things may have consisted of no more than RNA or RNA-like molecules bound to the surfaces of mineral particles. A key aspect of this theory is that these mineral particles have binding sites for RNA and its prebiotic precursors. The object of this study is to explore the binding properties of two of the best studied minerals, montmorillonite and hydroxylapatite, for possible precursors of RNA. The list of compounds investigated includes purines, pyrimidines, nucleosides, nucleotides, nucleotide coenzymes, diaminomaleonitrile and aminoimidazole carboxamide. Affinities for hydroxylapatite are dominated by ionic interactions between negatively charged small molecules and positively charged sites in the mineral. Binding to montmorillonite presents a more complex picture. These clay particles have a high affinity for organic ring structures which is augmented if they are positively charged. This binding probably takes place on the negatively charged faces of these sheet-like clay particles. Additional binding sites on the edges of these sheets have a moderate affinity for negatively charged molecules. Small molecules that bind to these minerals sometimes bind independently to sites on the minerals and sometimes bind cooperatively with favorable interactions between the bound molecules.

Adenine↗
PubMed1994

Clustering of low usage codons and ribosome movement.

A model is presented in which the distribution of low-usage codons in a message is a major factor in determining the impact that they will have on the translation rate and distribution of ribosomes on that message. This model is based on the assumption that low-usage codons are translated more slowly than normal codons, an assumption supported by various lines of published experimental evidence. Although the parameters used to develop this model are somewhat arbitrary, the main conclusions of this paper are consistent with a wide variation in the values of those parameters. In the model, low-usage codons arranged in clusters are much more effective in blocking ribosome movement on the message than ones that are dispersed. The effective size of the cluster is limited to the dimensions of the ribosome. It has been estimated that ribosomes on a message are spaced at least 27 nucleotides or nine codons apart. A ribosome translating a cluster of nine codons in which some or all of the codons are low-usage will move more slowly than over a comparable stretch of message containing no low-usage codons. Owing to ribosome size, the ribosome immediately behind the stalled ribosome will move as slowly; it must wait for the stalled ribosome to move on before it can even begin to translate the difficult region containing the low-usage codons. When the low-usage codon cluster is at the 3' end, the message will eventually be occupied by a ribosome jam that will transmit back to the 5' end of the message. In the steady state, the slowing effect imposed by a cluster of nine low-usage codons at the 3' end of a message would be just as great as if the entire message was composed of them. If the cluster is situated in the middle of a message, the ribosomes will form a jam upstream of the cluster. The ribosome density downstream of the cluster will be considerably reduced from what it would be for the same message with no cluster. If the cluster is at the 5' end of the message, the density of ribosomes will be reduced over the entire length of the message but the overall translation rate per ribosome will be only slightly reduced. However, owing to the reduced number of ribosomes initiating, the efficiency of the message in protein synthesis will be considerably reduced.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

Multicopy single-stranded DNAs with mismatched base pairs are mutagenic in Escherichia coli.

Retrons are genetic elements that encode multicopy single-stranded DNAs called msDNAs. They are clonally distributed in Escherichia coli and retrons in different clones produce DNAs with different nucleotide sequences. msDNAs consist of an RNA molecule covalently linked to a single-stranded DNA molecule. The latter contains an inverted repeat, resulting in a stem-loop structure. In two retrons, Ec83 and Ec78, the DNA is cleaved off from the RNA. All known retrons except Ec78, have one or more mismatched base pairs in the stem-loop structure. We found that two retrons, Ec86 and Ec83, when present in high copy numbers are mutagenic. The ratios of mutation frequencies observed in Lac- indicator strains were similar to the ratios observed for a mutant defective in mismatch repair. It is known that some proteins required for mismatch repair bind to mismatched base pairs prior to carrying out repair. The similarity in the mutation frequency ratios suggested that the mutagenesis caused by msDNAs of retrons Ec86 and Ec83 might be due to sequestration of a mismatch repair protein by msDNA. Strong support for this interpretation was obtained from the finding that the msDNA produced by retron Ec78 is not mutagenic.

Base Sequence↗
PubMed1993

Effects of consecutive AGG codons on translation in Escherichia coli, demonstrated with a versatile codon test system.

A system for testing the effects of specific codons on gene expression is described. Tandem test and control genes are contained in a transcription unit for bacteriophage T7 RNA polymerase in a multicopy plasmid, and nearly identical test and control mRNAs are generated from the primary transcript by RNase III cleavages. Their coding sequences, derived from T7 gene 9, are translated efficiently and have few low-usage codons of Escherichia coli. The upstream test gene contains a site for insertion of test codons, and the downstream control gene has a 45-codon deletion that allows test and control mRNAs and proteins to be separated by gel electrophoresis. Codons can be inserted among identical flanking codons after codon 13, 223, or 307 in codon test vectors pCT1, pCT2, and pCT3, respectively, the third site being six codons from the termination codon. The insertion of two to five consecutive AGG (low-usage) arginine codons selectively reduced the production of full-length test protein to extents that depended on the number of AGG codons, the site of insertion, and the amount of test mRNA. Production of aberrant proteins was also stimulated at high levels of mRNA. The effects occurred primarily at the translational level and were not produced by CGU (high-usage) arginine codons. Our results are consistent with the idea that sufficiently high levels of the AGG mRNA can cause essentially all of the tRNA(AGG) in the cell to become sequestered in translating peptidyl-tRNA(AGG) -mRNA-ribosome complexes stalled at the first of two consecutive AGG codons and that the approach of an upstream translating ribosome stimulates a stalled ribosome of frameshift, hop, or terminate translation.

Arginine↗
PubMed1991

Low-usage codons in Escherichia coli, yeast, fruit fly and primates.

Codon usage is compared between four classes of species, with an emphasis on characterization of low-usage codons. The classes of species analyzed include the bacterium Escherichia coli (ECO), the yeast Saccharomyces cerevisiae (YSC), the fruit fly Drosophila melanogaster (DRO), and several species of primates (PRI) (taken as a group; includes eleven species for which nucleotide sequence data have been reported to GenBank, however, greater than 90% of the sequences were from Homo sapiens). The number of protein-coding sequences analyzed were 968 for ECO, 484 for YSC, 244 for DRO, and 1518 for PRI. Three methods have been used to determine low-usage codons in these species. The first and most common way of assessing codon usage is by summing the number of time codons appear in reading frames of the genome in question. The second way is to examine the distribution of usage in different genes by scoring the number of protein reading frames in which a particular codon does not appear. The third way starts with a similar notion, but instead considers combinations of codons that are missing from the maximum number of genes. These three methods give very similar results. Each species has a unique combination of eight least-used codons, but all species contain the arginine codons, CGA and CGG. The agreement between YSC and PRI is particularly striking as they share six low-usage codons. All six carry the dinucleotide sequence, CG. The eight least-used codons in PRI include all codons that contain the CG dinucleotide sequence. Low-usage codons are clearly avoided in genes encoding abundant proteins for ECO, YSC DRO. In all species, proteins containing a high percentage of low-usage codons could be characterized as cases where an excess of the protein could be detrimental. Low codon usage is relatively insensitive to gross base composition. However, dinucleotide usage can sometimes influence codon usage. This is particularly notable in the case of CG dinucleotides in PRI.

Animals↗
PubMed1989

Two nearby sites bind zen protein independently.

The region upstream from the zerknullt (zen) gene contains three sites that specifically bind the zen protein product of the gene. Evidence for these binding sites was obtained by the filter binding technique and the DNase footprinting technique. The filter binding technique was used to scan various segments of DNA for the presence of possible specific binding sites. Segments that were selectively retained by the filter binding technique invariably contained one or more specific binding sites according to the DNase footprinting technique. Two of the zen protein binding sites were spaced only 30 base pairs apart. These sites could be separated without any loss in their specific binding properties. It is concluded that these two sites function independently in the binding of zen protein.

Animals↗
PubMed1988

Purification and properties of the Drosophila zen protein.

The zen protein is encoded by the zerknullt gene required for normal early development in Drosophila. Like many regulatory proteins of this type, zen contains a 60 amino acid homeobox sequence. We have purified the zen protein and studied its solution behavior and its interaction with DNA. The zen protein exists as a monomer in solution with a molecular weight of about 40,000. It binds specifically to a site about 900 bases upstream from the zen gene. Within this binding site DNase protection experiments indicate that binding is confined to two regions approximately 11 and 14 bases in length that are separated by about 30 base pairs. The protein concentration dependence of the binding curve suggests that protein binding is non cooperative.

Animals↗
PubMed1988

Regulation of gene expression in plasmid ColE1: delayed expression of the kil gene.

cea, imm, and kil are a cluster of three functionally related genes of the plasmid ColE1. The cea and kil genes are in the same inducible operon, with transcription being initiated from a promoter adjacent to the cea gene. The imm gene is located between the cea and kil genes, but it is transcribed in the opposite direction. Complementary interaction between the imm mRNA and the anti-imm sequences in the middle of the cea-kil transcript causes a pronounced delay in expression of the kil gene when the cea-kil operon is induced. A segment in the overlapping region between the cea and imm genes causes delayed expression of the kil gene in the absence of imm gene transcription. This delay effect increases the yields of colicin synthesized in induced cells.

Base Sequence↗
PubMed1985

Constancy of DNA organization of polymorphic and nonpolymorphic genes during development in Xenopus.

A study was undertaken to test for the occurrence of DNA rearrangements or amplifications during embryonic development in Xenopus laevis. DNA isolated from testes and liver was digested with four restriction enzymes, separated on agarose gels, transferred to nitrocellulose, and hybridized with over 50 cloned cDNA probes generated from embryonic poly (A)+ RNA. No qualitative or quantitative differences were detectable in the DNA hybridization patterns of testes and liver DNA, suggesting that, at least during liver development, selective amplifications or rearrangements occur rarely if at all. In the course of this investigation a wide range of restriction-site polymorphisms for different genes was observed. While some genes showed little polymorphism among different animals, several genes showed considerable polymorphism, involving changes in several restriction enzyme sites. These complex polymorphisms could be the result of gene rearrangements that occur occasionally during the course of sexual reproduction rather than during development.

Aging↗
PubMed1985

Colicin synthesis and cell death.

Colicin E1 is a small plasmid, containing the cea gene for colicin, the most prominent product of the plasmid. Colicin is a 56-kilodalton bacteriocin which is especially toxic to Escherichia coli cells that do not contain the plasmid. Under normal growth conditions very low levels of the plasmid are produced as a result of cea gene repression by the host LexA protein. Conditions that lower the concentration of LexA protein result in elevated levels of colicin synthesis. The LexA protein concentration can be lowered by exposing the cells to DNA-damaging reagents such as UV light or mitomycin C. This is because DNA damage signals the host SOS response; the response leads to activation of the RecA protease which degrades the LexA protein. DNA-damaging reagents result in very high levels of colicin synthesis and subsequent death of plasmid-bearing cells. Elevated levels of colicin are also produced in mutants of E. coli that are deficient in LexA protein. We found that comparably high levels of colicin can be produced in such mutants in the absence of cell death. In lexA strains carrying a defective LexA repressor, colicin synthesis shows a strong temperature dependence. Ten to twenty times more colicin is synthesized at 42 degrees C. This sharp dependence of synthesis on temperature suggests that there are factors other than the LexA protein which regulate colicin synthesis.

Bacterial Proteins↗
PubMed1985

Mitomycin-induced lethality of Escherichia coli cells containing the ColE1 Plasmid: involvement of the kil gene.

Escherichia coli cells containing the ColE1 plasmid or related plasmids are killed by considerably lower levels of mitomycin C (MTC) than are plasmid-free cells. Since exposure to MTC induces high levels of synthesis of the plasmid-encoded colicin toxin, it was originally thought that the killing effect was due to the increased levels of colicin. This possibility was discounted when it was shown that deletion mutations in the plasmid lacking most of the colicin (cea) gene still sensitized host cells to MTC. Only when the region containing the cea gene promoter was deleted did the killing effect disappear. This led to the suggestion that transcription originating from the cea gene promoter and not the colicin protein itself was required for killing. Transcription-blocking mutations in the cea gene support this suggestion. It was proposed that there is a gene (kil) located downstream from the cea gene in the same operon that is responsible for MTC killing and colicin transport. The precise location of the kil gene in ColE1 can be predicted by piecing together published sequence information. We used available sequence data to construct a number of well-defined plasmid mutants to further examine the relevance of transcription from the cea promoter and the kil gene to drug-induced killing and colicin transport. The most informative mutant had a small insertion in the kil gene. This mutant behaved as predicted; cells containing it had a greatly lowered sensitivity to MTC and were severely inhibited in the transport of colicin.

Bacteriocin Plasmids↗
PubMed1983

Analysis of ColE1 expression in vitro after chromosome fragmentation.

The RNA and protein products synthesized from ColE1 DNA were observed before and after cutting the DNA with different restriction enzymes. Synthesis was carried out in the DNA-directed coupled transcription translation system. The S-30 extracts used to catalyze synthesis were prepared from a recB mutant in which the linear DNA fragments resulting from restriction enzyme cleavage were spared from the usual degradation by exonucleolytic attack. By correlating the observed in vitro synthesized products with the location of the cleavage sites in the plasmid chromosome, it was possible to identify specific gene products. The col gene catalyzes the synthesis of numerous peptides in addition to the 56-kilodalton colicin protein encoded by this gene. Most of the subsidiary products appear to arise as the result of premature termination by a mechanism(s) which remains to be determined. A unique RNA and protein were characterized as products of the imm gene. The RNA has an estimated mass of 150 kilodaltons, and the protein has an estimated mass of 13 kilodaltons. From the DNA sequence of the chromosome, it was concluded that the transcripts from the imm and col genes must crisscross each other over a region of about 75 base pairs. Such a pattern of transcription might lead to interference of transcription of one gene by the other gene. Consistent with this hypothesis, it was found that imm gene transcription increased severalfold in vitro when the chromosome was cleaved in a way that eliminated transcription originating at the col gene promoter. Surprisingly, the increase in transcription by this mechanism did not result in a significant increase in the synthesis of the imm gene-encoded protein.

Bacterial Proteins↗
PubMed1982

Resistance of adenoviral DNA replication to aphidicolin is dependent on the 72-kilodalton DNA-binding protein.

Aphidicolin is a highly specific inhibitor of DNA polymerase alpha and has been most useful for assessing the role of this enzyme in various replication processes (J. A. Huberman, Cell 23:647-648, 1981). Both nuclear DNA replication and simian virus 40 DNA replication are highly sensitive to this drug (Krokan et al., Biochemistry 18:4431-4443, 1979), whereas mitochondrial DNA synthesis is completely insensitive (Zimmerman et al., J. Biol. Chem. 255:11847-11852, 1980). Adenovirus DNA replication is sensitive to aphidicolin, but only at much higher concentrations. These patterns of sensitivity are seen both in vivo and in vitro (Krokan et al., Biochemistry 18:4431-4443, 1979). A temperature-sensitive mutant of adenovirus type 5 known as H5ts125 is able to complete but not initiate new rounds of replication at nonpermissive temperatures (P. C. van der Vliet and J. S. Sussenbach, Virology 67:415-426, 1975). When cells infected with H5ts125 were shifted from permissive (33 degrees C) to nonpermissive (41 degrees C) conditions, the residual DNA synthesis (elongation) showed a striking increase in sensitivity to aphidicolin. The temperature-sensitive mutation of H5ts125 is in the gene for the 72-kilodalton single-stranded DNA-binding protein. This demonstrated that the increased resistance to aphidicolin shown by adenovirus DNA replication was dependent on that protein. It also supports an elongation role for both DNA polymerase alpha and the 72-kilodalton single-stranded DNA-binding protein in adenovirus DNA replication. Further support for an elongation role of DNA polymerase alpha came from experiments with permissive temperature conditions and inhibiting levels of aphidicolin in which it was shown that newly initiated strands failed to elongate to completion.

Adenoviruses, Human↗