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At least 181 records · Page 10Linked to original sources
PubMed1991

The tdh and serA operons of Escherichia coli: mutational analysis of the regulatory elements of leucine-responsive genes.

The tdh promoter of Escherichia coli is induced seven- to eightfold when cells are grown in the presence of exogenous leucine. A scheme was devised to select mutants that exhibited high constitutive expression of the tdh promoter. The mutations in these strains were shown to lie within a previously identified gene (lrp) that encodes Lrp (leucine-responsive regulatory protein). By deletion analysis, the site of action of Lrp was localized to a 25-bp region between coordinates -69 and -44 of the tdh promoter. Disruption of a 12-bp presumptive target sequence found in this region of tdh resulted in constitutively derepressed expression from the tdh promoter. Similar DNA segments (consensus, TTTATTCtNaAT) were also identified in a number of other promoters, including each of the Lrp-regulated promoters whose nucleotide sequence is known. The sequence of the promoter region of serA, an Lrp-regulated gene, was determined. No Lrp consensus target sequence was present upstream of serA, suggesting that Lrp acts indirectly on the serA promoter. A previously described mutation in a leucine-responsive trans-acting factor, LivR (J. J. Anderson, S. C. Quay, and D. L. Oxender, J. Bacteriol. 126:80-90, 1976), resulted in constitutively repressed expression from the tdh promoter and constitutively induced expression from the serA promoter. The possibility that LivR and Lrp are allelic is discussed.

Alcohol Oxidoreductases↗
PubMed1989

Gene replacement and retrieval with recombinant M13mp bacteriophages.

We have developed an allele exchange system for shuttling sequences of DNA to and from their original chromosomal loci. Cloned segments of the histidine operon of Salmonella typhimurium and the lactose operon of Escherichia coli served as target sequences and were used to develop the system. Replacement and retrieval of target sequences used the phage M13mp vectors and proceeded through an M13 lysogen intermediate. The intermediates and products of allele exchange were characterized by genetic and hybridization analyses. Several unique properties of M13 lysogens were exploited to devise positive selections to detect integration and excision. These positive selections were used to manipulate phenotypically silent alleles.

Chromosome Deletion↗
PubMed1989

Conservation of complex DNA recognition domains between families of restriction enzymes.

One polypeptide, designated S, confers sequence-specificity to the multisubunit type I restriction enzymes. Two families of such enzymes, K and A, include members that recognize diverse, bipartite, target sequences. The S polypeptides of the K family, while having areas of near identity, also contain two extensive regions of variable sequence. We now show that one of these, comprising the N-terminal 150 amino acids, specifies recognition of one component of the bipartite target sequence. We have determined the sequence recognized by EcoE, a member of the A family. This sequence, 5'GAG(N7)ATGC, has the trinucleotide GAG in common with EcoA and with StySB of the K family. We determined the nucleotide sequences of the S genes of EcoA and EcoE, and compared their predicted amino acid sequences with each other and with those of the five members of the K family. There is no general sequence similarity between families, but the domain of the S polypeptide of StySB, which specifies GAG, shows nearly 50 per cent identity with the amino variable region of the S polypeptides of EcoA and EcoE. A complex domain that recognizes and directs methylation of GAG is therefore common to enzymes of generally dissimilar amino acid sequence.

Amino Acid Sequence↗
PubMed1991

Blocking of Tat-dependent HIV-1 RNA modification by an inhibitor of RNA polymerase II processivity.

Human immunodeficiency virus gene expression is regulated transcriptionally and post-transcriptionally by the virally encoded tat protein (Tat). Tat functions through an RNA target sequence located in the untranslated region at the 5' end of viral transcripts. In Xenopus oocytes, translation of RNA containing the target sequence is specifically activated by Tat. This activation only occurs if the RNA is injected into the nucleus, and might be due to Tat-dependent, nucleus-specific chemical modification of the RNA which somehow facilitates translation. Here we demonstrate that Tat activation of its target RNA in the nucleus involves a Tat-dependent covalent modification. The modified RNA is competent for translation after reinjection into either the nucleus or the cytoplasm in the absence of Tat. Furthermore, we find that the nucleoside analogue 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, which inhibits processivity of RNA polymerase II (ref. 9), blocks this Tat-dependent modification.

Animals↗
PubMed1988

The effects of variations in the number and sequence of targeting signals on nuclear uptake.

To determine if the number of targeting signals affects the transport of proteins into the nucleus, Xenopus oocytes were injected with colloidal gold particles, ranging in diameter from 20 to 280 A, that were coated with BSA cross-linked with synthetic peptides containing the SV-40 large T-antigen nuclear transport signal. Three BSA conjugate preparations were used; they had an average of 5, 8, and 11 signals per molecule of carrier protein. In addition, large T-antigen, which contains one signal per monomer, was used as a coating agent. The cells were fixed at various times after injection and subsequently analyzed by electron microscopy. Gold particles coated with proteins containing the SV-40 signal entered the nucleus through central channels located within the nuclear pores. Analysis of the intracellular distribution and size of the tracers that entered the nucleus indicated that the number of signals per molecule affect both the relative uptake of particles and the functional size of the channels available for translocation. In control experiments, gold particles coated with BSA or BSA conjugated with inactive peptides similar to the SV-40 transport signal were virtually excluded from the nucleus. Gold particles coated with nucleoplasmin, an endogenous karyophilic protein that contains five targeting signals per molecule, was transported through the nuclear pores more effectively than any of the BSA-peptide conjugates. Based on a correlation between the peri-envelope density of gold particles and their relative uptake, it is suggested that the differences in the activity of the two targeting signals is related to their binding affinity for envelope receptors. It was also determined, by performing coinjection experiments, that individual pores are capable of recognizing and transporting proteins that contain different nuclear targeting signals.

Amino Acid Sequence↗
PubMed1990

2'-O-alkyl oligoribonucleotides as antisense probes.

2'-O-Methyl oligoribonucleotides have recently been introduced as antisense probes for studying RNA processing and for affinity purification of RNA-protein complexes. To identify RNA analogues with improved properties for antisense analysis, 2'-O-alkyl oligoribonucleotides were synthesized in which the alkyl moiety was either the three-carbon linear allyl group or the five-carbon branched 3,3-dimethylallyl group. Both these analogues were found to be completely resistant to degradation by either DNA- or RNA-specific nucleases. Use of biotinylated derivatives of the probes to affinity-select ribonucleoprotein particles from crude HeLa cell nuclear extracts showed that the presence of the bulky 3,3-dimethylallyl group significantly reduces affinity selection, whereas the allyl derivative binds rapidly and stably to targeted sequences and affinity-selects efficiently. The allyl derivatives also showed an increase in the level of specific binding to targeted sequences compared with 2'-O-methyl probes of identical sequence. These properties indicate that the 2'-O-allyl oligoribonucleotides are particularly well suited for use as antisense probes.

Alkylation↗
PubMed1991

Amplification of nucleic acids by polymerase chain reaction (PCR) and other methods and their applications.

The in vitro replication of DNA, principally using the polymerase chain reaction (PCR), permits the amplification of defined sequences of DNA. By exponentially amplifying a target sequence, PCR significantly enhances the probability of detecting target gene sequences in complex mixtures of DNA. It also facilitates the cloning and sequencing of genes. Amplification of DNA by PCR and other newly developed methods has been applied in many areas of biological research, including molecular biology, biotechnology, and medicine, permitting studies that were not possible before. Nucleic acid amplification has added a new and revolutionary dimension to molecular biology. This review examines PCR and other in vitro nucleic acid amplification methodologies--examining the critical parameters and variations and their widespread applications--giving the strengths and limitations of these methodologies.

Bacterial Infections↗
PubMed1988

Theoretical analysis of 'addressed' chemical modification of DNA.

Chemical "addressed" modification of DNA involves treatment of single-stranded DNA with oligonucleotides complementary to certain target sequences in this DNA and bearing a groupings reactive towards DNA bases. The binding of oligonucleotides can occur both at completely (specific) and incompletely (nonspecific) complementary sites. We analyse the modification of a fragment that is flanked by two target sequences complementary to a given oligonucleotide address, contains no more such targets and has some randomly distributed sites for nonspecific binding. Conditions for the maximum ratio between specific and non-specific modification are determined. We find the probability of both target termini being specifically modified without any non-specific modification occurring within the fragment up to a given moment in time. Quantitative analysis is based on the use of known features of the specific and non-specific binding of an oligonucleotide to DNA sites. This analysis shows the possibility of specific cutting of DNA based on addressed modification.

Base Composition↗
PubMed1990

Simultaneous detection of different mRNA sequences coding for neuropeptide hormones by double in situ hybridization using FITC- and biotin-labeled oligonucleotides.

Oligonucleotides labeled with FITC or biotin were applied for detection of specific mRNAs in microscopic preparations by in situ hybridization. The oligonucleotides were labeled with one FITC or biotin molecule at the 5' end or with a tail of biotin molecules at the 3' end. The target sequences were mRNAs coding for an ovulation hormone (CDCH) in the caudodorsal cells (CDC) of the pond snail Lymnaea stagnalis and a molluscan insulin-like peptide (MIP) in the light green cells (LGC) of the same organism. The hybridized oligonucleotides were detected either directly after the hybridization procedure by fluorescence microscopy or indirectly after an immunocytochemical procedure to visualize the biotin or FITC moiety. The results indicate that the detectability of the mRNA sequences is at least partially dependent on the accessibility of the target sequences for the immunocytochemical detection systems. The positive hybridization results obtained with oligonucleotides containing different labels enabled us to perform double hybridization experiments for simultaneous detection of CDCH and MIP mRNAs in one tissue section. Using FITC- and biotin-labeled oligonucleotides, we also demonstrated simultaneously different sequences on the same mRNA molecule.

Animals↗
PubMed1989

Determinant differences between the rabbit and mouse immunoglobulin kappa enhancers impair the activity of the rabbit enhancer in mouse myeloma cells.

Enhancer activity of the rabbit immunoglobulin kappa light chain gene intron conserved region (KICR) was examined in mouse myeloma cells using transient expression experiments. Compared to the homologous region of the mouse kappa light chain gene, the rabbit KICR shows nearly no stimulatory effect on expression of the indicator gene, cat. Experiments with mouse-rabbit chimeric KICRs indicated that differences in the region around the NF-kappa B binding site are responsible for the impaired activity of the rabbit KICR whereas mouse sequences covering the kappa E2 and kappa E3 motifs can be replaced by the equivalent rabbit fragment without affecting enhancer function. Creation of a perfect mouse NF-kappa B target sequence in the rabbit gene only partially restores enhancer activity. Furthermore, mouse and rabbit DNA fragments encompassing the NF-kappa B target sequence behave in an identical manner in an electrophoretic mobility shift assay. The results indicate species-related functional differences in the immunoglobulin kappa light chain gene enhancer and suggest that although the NF-kappa B binding site plays a crucial role in enhancer activity surrounding gene elements are also necessary for full enhancer effect.

Animals↗
PubMed1988

Sequence analysis of termini of conjugative transposon Tn916.

Transposon Tn916 is a 16.4-kilobase, broad-host-range, conjugative transposon originally identified on the chromosome of Enterococcus (Streptococcus) faecalis DS16. Its termini have been sequenced along with the junction regions for two different insertions. The ends were found to contain imperfect inverted repeat sequences with identity at 20 of 26 nucleotides. Further in from the ends, imperfect directly repeated sequences were present, with 24 of 27 nucleotides matching. The transposon junction regions contained homologous segments but of a nature not consistent with a direct duplication of the target sequence. Within the right terminus was a potential outwardly reading promoter. Tn916 is believed to transpose via an excision-insertion mechanism; based on the analyses of the termini, as well as two target sequences (before insertion and after excision), a possible model is suggested.

Base Sequence↗
PubMed1986

Yeast mitochondrial ATPase subunit 8, normally a mitochondrial gene product, expressed in vitro and imported back into the organelle.

Subunit 8 of yeast mitochondrial F1F0-ATPase is a proteolipid made on mitochondrial ribosomes and inserted directly into the inner membrane for assembly with the other F0 membrane-sector components. We have investigated the possibility of expressing this extremely hydrophobic, mitochondrially encoded protein outside the organelle and directing its import back into mitochondria using a suitable N-terminal targeting presequence. This report describes the successful import in vitro of ATPase subunit 8 proteolipid into yeast mitochondria when fused to the targeting sequence derived from the precursor of Neurospora crassa ATPase subunit 9. The predicted cleavage site of matrix protease was correctly recognized in the fusion protein. A targeting sequence from the precursor of yeast cytochrome oxidase subunit VI was unable to direct the subunit 8 proteolipid into mitochondria. The proteolipid subunit 8 exhibited a strong tendency to embed itself in mitochondrial membranes, which interfered with its ability to be properly imported when part of a synthetic precursor.

Biological Evolution↗
PubMed1990

A novel pathway for secretory proteins?

In eukaryotes, most proteins which are transported to the extracellular space, into mitochondria or into chloroplasts are synthesized as precursor polypeptides containing cleavable N-terminal signal or targeting sequences. We have searched the literature for proteins that are exported from the cytosol without being proteolytically processed. Some of these proteins contain uncleaved signal or targeting sequences. However, among secretory proteins there is a class that does not possess hydrophobic signal sequences and appears to leave the cell by a secretory pathway clearly distinct from the classical route through the endoplasmic reticulum and Golgi apparatus.

Acylation↗
PubMed1992

Sexing of mouse preimplantation embryos by detection of Y chromosome-specific sequences using polymerase chain reaction.

Detection of genes known to be present on the mammalian Y chromosome was adapted for sexing mouse early embryos using the polymerase chain reaction (PCR) method. Sry and Zfy genes located in the sex-determining region of the Y chromosome were chosen for Y-specific target sequences, and DXNds3 sequence on the X chromosome was chosen for control. The two-step PCR method using two pairs of primers for each of the target sequences was employed for detecting the sequences. When DNAs of male and female mice were amplified with these primers, male-specific fragments were detected even in DNAs that were equivalent in amount to two cells. Mouse embryos at the two-cell stage were separated into two individual blastomeres, and one blastomere was karyotyped at the second cleavage. The remaining blastomere was subjected to PCR amplification immediately or after having been cultured for 48 h up to the morula stage. The Sry and Zfy sequences were detected in about half the embryos; detection of the Sry and Zfy sequences corresponded exactly to the presence of the Y chromosome, except in one sample of male morula in which embryos may have been lost before the PCR amplification. It is concluded that the sex of mouse preimplantation embryos can be accurately determined through detection of the Y-specific sequences using the two-step PCR method, even with the single blastomeres separated at the two-cell stage.

Animals↗
PubMed1991

An improved method for detection of B-lymphoid clonality by polymerase chain reaction.

Several groups have recently described methods for the detection of clonal immunoglobulin heavy chain (IgH) gene rearrangements in B-cell malignancies by polymerase chain reaction (PCR) gene amplification using variable region-(VH) and joining (JH) region-specific primers. The simplest methods utilize a single VH primer specific for sequences present in most VH regions corresponding to the third framework region (FR3). An alternative approach is to use a panel of VH family-specific primers specific for the first framework regions (FR1). In the course of nucleotide sequence analysis of IgH gene rearrangements amplified using a VH FR1 primer panel, these authors previously observed 3' VH region deletion and/or base mis-matches sufficient to prevent efficient priming from the VH FR3 primer target sequence in a significant minority of cases of B-lineage malignancy. An improved PCR method has therefore been developed by using a panel of seven VH FR1 family-specific primers incorporated in a single reaction. By using this method clonal IgH gene rearrangement is detected in 15 of 16 cases of B-lineage malignancy. Significantly, this series included four cases of B-lymphoma in which previous attempts to detect PCR clonal IgH gene rearrangements using a VH FR3 primer were unsuccessful. In two of these cases, nucleotide sequence analysis of the amplified DNA showed that failure to prime with the VH FR3 primer was likely to be attributable to insufficient homology with the target sequence. The use of the approach described in this paper should significantly improve the reliability of detection of B-lymphoid clonality by PCR.

Base Sequence↗
PubMed1990

Polymerase chain reaction amplification of pseudorabies virus DNA from acutely and latently infected cells.

A characteristic of alphaherpesviruses, including pseudorabies virus (PRV), is that the acute phase of the disease is followed by lifelong latency. Latently infected animals are asymptomatic but can transmit reactivated virus. Corticosteroid administration, tissue explanation, blot- and in situ hybridizations have been used to demonstrate the presence of latent PRV infections. The use of blot hybridization as a convenient method for defining the incidence of PRV infections in swine herds has been hampered by the detection limit of this method. The objective of this study was to increase this sensitivity of blot hybridization by polymerase chain reaction (PCR) amplification of target sequences. Two sets of 20-mer primers were synthesized and used to amplify gX and gII glycoprotein gene sequences in two different strains of PRV. The specificity of the amplification was verified by Southern blot hybridization and restriction endonuclease analysis of the amplified fragments. Amplification of target sequences by PRC increased their detection limit by a factor of at least 10(5). Porcine ganglion samples, in which latency had been demonstrated by in vitro explanation, were analyzed by PCR together with positive and negative controls. Duplicate slot blot analyses of a portion of the amplified products were used to demonstrate latency in seven of eight samples. It was concluded that blot hybridization of PCR amplified DNA appears to be both a sensitive and convenient method for the detection of PRV induced latency.

Animals↗
PubMed1991

Polymerase chain reaction and Q beta replicase amplification.

The polymerase chain reaction (PCR) and Q beta replicase are two methods in which nucleic acid polymerases are used for amplification. Although these approaches share many similar problems concerning target contamination and probe specificity, they differ dramatically in their mechanisms of action and modes of application. The PCR method amplifies target sequences between two priming oligonucleotides and in essence amplifies a portion of the analyte. Q beta replicase, on the other hand, amplifies a specific template molecule hybridized to target sequences and therefore amplifies a signal component of the system. For this reason, Q beta replicase amplification has applications in areas other than for the detection of nucleic acid sequences. The requirements for application and the advantages of both PCR and Q beta replicase amplification are reviewed.

DNA-Directed DNA Polymerase↗
PubMed1986

FLP site-specific recombinase of yeast 2-micron plasmid. Topological features of the reaction.

The 2-micron plasmid of the yeast Saccharomyces cerevisiae encodes a site-specific recombinase (FLP) that promotes inversion across a unique site contained in each of the 599-base-pair inverted repeats of the plasmid. We have studied the topological changes generated in supercoiled substrates after exposure to the purified FLP protein in vitro. When a supercoiled substrate bearing two FLP target sequences in inverse orientation is treated with FLP, the products are multiply knotted structures that arise as a result of random entrapment of interdomainal supercoils. Likewise, a supercoiled substrate bearing two target sequences in direct orientation yields multiply interlocked catenanes as the product. Both types of substrate seem to be able to undergo repeated rounds of recombination that result in products of further complexity. The FLP protein also acts as a site-specific topoisomerase during the recombination reaction.

Binding Sites↗