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R F Gesteland

Publications and source records attributed to R F Gesteland.

At least 19 recordsLinked to original sources
PubMed1992

Digital chemiluminescence imaging of DNA sequencing blots using a charge-coupled device camera.

Digital chemiluminescence imaging with a cryogenically cooled charge-coupled device (CCD) camera is used to visualize DNA sequencing fragments covalently bound to a blotting membrane. The detection is based on DNA hybridization with an alkaline phosphatase(AP) labeled oligodeoxyribonucleotide probe and AP triggered chemiluminescence of the substrate 3-(2'-spiro-adamantane)-4-methoxy-4-(3"-phosphoryloxy)phenyl- 1,2-dioxetane (AMPPD). The detection using a direct AP-oligonucleotide conjugate is compared to the secondary detection of biotinylated oligonucleotides with respect to their sensitivity and nonspecific binding to the nylon membrane by quantitative imaging. Using the direct oligonucleotide-AP conjugate as a hybridization probe, sub-attomol (0.5 pg of 2.7 kb pUC plasmid DNA) quantities of membrane bound DNA are detectable with 30 min CCD exposures. Detection using the biotinylated probe in combination with streptavidin-AP was found to be background limited by nonspecific binding of streptavidin-AP and the oligo(biotin-11-dUTP) label in equal proportions. In contrast, the nonspecific background of AP-labeled oligonucleotide is indistinguishable from that seen with 5'-32P-label, in that respect making AP an ideal enzymatic label. The effect of hybridization time, probe concentration, and presence of luminescence enhancers on the detection of plasmid DNA were investigated.

Adamantane↗
PubMed1992

Seven, eight and nine-membered anticodon loop mutants of tRNA(2Arg) which cause +1 frameshifting. Tolerance of DHU arm and other secondary mutations.

The mutant tRNA(2Arg) encoded by the genetically-selected frameshift suppressor, sufT621, inserts arginine and causes a +1 reading-frame shift at the proline codon, CCG(U). There is an extra base, G36.1, in argV beta, one of the four identical genes for tRNA(2Arg) in the position between bases 36 and 37, corresponding to the 3' side of the anticodon. The new four-base anticodon, predicted from DNA sequencing to be 3' GGCA 5', is complementary to the four-base codon CCGU. Quadruplet translocation promoted by mutant argV does not require perfect complementarity between the codon and the anticodon since synthetic genes encoding derivatives of tRNA(2Arg) and tRNA(1Pro), with four-base anticodons complementary to three out of the four bases of CCGU, were also shown to be capable of frameshifting. Two other mutants of argV, inferred to have normal-size, seven-base anticodon loops, were also found to be capable of four-base-decoding demonstrating that quadruplet translocation promoted by mutant argV does not require an enlarged anticodon loop. Other alleles of argV, predicted to have nine bases in the anticodon loop, were also found to cause frameshifting. The DNA sequence of two of these showed in addition, either a deletion of G24, or a ten-base duplication in the region corresponding to the TFC arm. A general finding is that mutations in the DHU arm of tRNA(2Arg) are compatible with, and in one case necessary for, frameshifting.

Amino Acid Sequence↗
PubMed1992

Low fluorescence background electroblotting membrane for DNA sequencing.

A low fluorescence background polypropylene (PP) membrane has been developed for ultimate use as an electroblotting membrane in DNA sequencing based on fluorescence detection. The DNA binding capacity of this membrane is improved by a surface modification using radio frequency plasma discharge (RFPD) in ammonia gas. The RFPD operational parameters are evaluated both in terms of membrane nitrogen content and in terms of the product's capacity for binding radioisotope-labeled DNA fragments. The surface morphologies of the derivatized membranes are examined by scanning electron microscopy; their mechanical and electrical properties, which are important for the subsequent sequencing procedures, are likewise established. Due to the goal of developing a membrane suitable for multiplex processing, in which the electroblotted DNA must withstand dozens of hybridization/stripping cycles, special attention is given the covalent attachment of DNA to the membrane. The modified PP membrane is evaluated in a multiplex sequencing application using radioisotope-labeled DNA probes, and found to yield somewhat better binding of a given amount of electroblotted DNA than the commonly used GeneScreen membrane. A tenfold repetition of the probing indicates little loss of signal; the membrane-bound DNA is stable upon storage and shows no detectable loss in probing efficiency after one month.

Base Sequence↗
PubMed1991

An analysis of sequences stimulating frameshifting in the decoding of gene 10 of bacteriophage T7.

The signals necessary for the translational frameshift in the gene 10 message of bacteriophage T7 include the previously identified frameshift site and the 3' non-coding region, over 200 bases downstream. The functional components of the frameshift site are identified in this study and show that the site most probably operates by the retroviral type two site mechanism. However, the base pairing requirements for the first tRNA are much more relaxed after the slip than is seen in other examples. The element at the 3' end of the gene, also necessary for frameshifting, is examined but only the extreme 5' side of the transcriptional terminator stem-loop structure in the 3' non-coding region seems to be required. No simple secondary structural model can explain the involvement of this sequence. The T7 frameshift site can be replaced with either a T3 site or a E. coli dnaX site. Both show higher levels of frameshifting than with the T7 site.

Base Sequence↗
PubMed1991

Multiwavelength fluorescence detection for DNA sequencing using capillary electrophoresis.

Multiwavelength detection of laser induced fluorescence for dideoxynucleotide DNA sequencing with four different fluorophores and separation by capillary gel electrophoresis is described. A cryogenically cooled, low readout noise, 2-dimensional charge-coupled device is used as a detector for the on-line, on-column recording of emission spectra. The detection system has no moving parts and provides wavelength selectivity on a single detector device. The detection limit of fluorescently labeled oligonucleotides meets the high sensitivity requirements for capillary DNA sequencing largely due to the efficient operation of the CCD detector with a 94% duty cycle. Using the condition number as a selectivity criterion, multiwavelength detection provides better analytical selectivity than detection with four bandpass filters. Monte Carlo studies and analytical estimates show that base assignment errors are reduced with peak identification based on entire emission spectra. High-speed separation of sequencing samples and the treatment of the 2-dimensional electropherogram data is presented. Comparing the DNA sequence of a sample separated by slab gel electrophoresis with sequence from capillary gel electrophoresis and multiwavelength detection we find no significant difference in the amount of error attributable to the instrumentation.

Base Sequence↗
PubMed1991

Evidence that a downstream pseudoknot is required for translational read-through of the Moloney murine leukemia virus gag stop codon.

Approximately 5% of the ribosomes translating the gag gene of murine leukemia viruses read through the UAG terminator and translate the in-frame pol gene to produce the gag-pol fusion polyprotein, the sole source of the pol gene products. We show that a pseudoknot located eight nucleotides 3' of the UAG codon in the Moloney murine leukemia virus is required for read-through. This requirement is markedly different from that known to be involved in other cases of read-through but surprisingly similar to some stimulatory sequences known to promote ribosomal frameshifting.

Animals↗
PubMed1991

The signal for a leaky UAG stop codon in several plant viruses includes the two downstream codons.

Expression of the RNA replicase domain of tobacco mosaic virus (TMV) and certain protein-coding regions in other plant viruses, is mediated by translational readthrough of a leaky UAG stop codon. It has been proposed that normal tobacco tyrosine tRNAs are able to read the UAG codon of TMV by non-conventional base-pairing but recent findings that stop codons can also be bypassed as a result of extended translocational shifts (tRNA hopping) have encouraged a re-examination. In light of the alternatives, we investigated the sequences flanking the leaky UAG codon using an in vivo assay in which bypass of the stop codon is coupled to the transient expression of beta-glucuronidase (GUS) reporter genes in tobacco protoplasts. Analysis of GUS constructions in which codons flanking the stop were altered allowed definition of the minimal sequence required for read through as UAG-CAA-UUA. The effects of all possible single-base mutations in the codons flanking the stop indicated that 3' contexts of the form CAR-YYA confer leakiness and that the 3' context permits read through of UAA and UGA stop codons as well as UAG. Our studies demonstrate a major role for the 3' context in the read through process and do not support a model in which teh UAG is bypassed exclusively as a result of anticodon-codon interactions. No evidence for tRNA hopping was obtained. The 3' context apparently represents a unique sequence element that affects translation termination.

Base Composition↗
PubMed1991

Frameshifting in gene 10 of bacteriophage T7.

Gene 10 of bacteriophage T7, which encodes the most abundant capsid protein, has two products: a major product, 10A (36 kDa), and a minor product, 10B (41 kDa). 10B is produced by frameshifting into the -1 frame near the end of the 10A coding frame and is incorporated into the capsid. The frameshift occurs at a frequency of about 10% and is conserved in bacteriophage T3. This study shows that sequences important to frameshifting include the originally proposed frameshift site, consisting of overlapping phenylalanine codons and the 3' noncoding region that includes the transcriptional terminator over 200 bases downstream of the frameshift site. The frameshift occurs at the overlapping phenylalanine codons as determined from peptide sequencing data. Complementation studies show that there is only a very weak phenotype associated with phage infections in which there is no 10A frameshifting. Capsids from such infections are devoid of 10B and are as stable as wild-type capsids.

Amino Acid Sequence↗
PubMed1990

The role of EF-Tu and other translation components in determining translocation step size.

The two EF-Tu encoding genes, tufA and tufB, of Salmonella typhimurium have been sequenced. Nearly all the differences from their Escherichia coli counterparts are third position changes which do not alter the encoded amino acids. Unexpectedly, most of the changes in one Salmonella tuf gene are paralleled by changes in the other tuf gene perhaps due to gene repair despite the distance separating the genes. Three mutants which cause mis-framing, have their substitutions at codon 375. Explanations for mutants which cause mis-framing are considered and the mechanism of normal reading frame maintenance discussed.

Escherichia coli↗
PubMed1990

Transcriptional slippage occurs during elongation at runs of adenine or thymine in Escherichia coli.

A run of 11 adenine or thymine residues at the 5' end of an out-of-frame lacZ gene causes a high level of beta-galactosidase expression in E. coli. This effect was not observed for a run of guanine residues. Reverse transcription of mRNA isolated from E. coli containing the run of 11 A's reveals heterogeneity of transcript length while reverse transcription of mRNA isolated from S. cerevisiae containing the same gene shows no heterogeneity. Protein sequencing of the beta-galactosidase molecules derived from the out-of-frame construct containing a run of adenines reveals the addition of a lysine at the run. A new method was developed where messages small enough to allow resolution of single nucleotide differences on an acrylamide gel are electrophoresed, electroblotted onto nylon and probed. This confirmed the reverse transcription results and showed that additional residues can be added to transcripts derived from DNA containing 10 or 11 thymine residues. A mechanism for slippage is discussed where the A-U rich RNA-DNA hybrid can denature during elongation and rehybridize in an offset position, causing the addition of extra residues to the transcript.

Adenine↗
PubMed1990

Saturation mutagenesis of a DNA region of bend. Base steps other than ApA influence the bend.

A 60 base-pair region of a simian virus 40 DNA fragment was mutagenized to determine base-pairs that are critical for the fragment to bend. The site-directed mutagenesis saturated this region with all possible single base-pair substitutions. The mobility of each mutated fragment was measured by polyacrylamide electrophoresis at 4 degrees C and at 65 degrees C to assess the degree of bend. Four conclusions can be drawn. First, interruptions within the A tracts and changes in the phasing of the A tracts alter the degree of bend. Second, G tracts phased at a half-helical turn from an A tract are additive to the bend. Third, guanine residues in a nearest-neighbor contact with the A tracts modify the bend. Fourth, some mutations that do not obviously relate to the A tracts also alter the DNA bend and suggest clearly that base steps other than ApA are involved in sequence-directed DNA bends.

Base Composition↗
PubMed1990

Guanine tracts enhance sequence directed DNA bends.

Synthetic DNA fragments were constructed to determine the effect of G tracts, in conjunction with periodically spaced A tracts, on DNA bends. Relative length measurements showed that the G tracts spaced at the half helical turn enhanced the DNA bend. When the G tract was interrupted with a thymine or shortened to one or two guanines, the relative lengths decreased. If the G tract was replaced with either an A tract or a T tract, the bend was cancelled. Replacement with a C tract decreased the relative length to that of a thymine interruption suggesting that bend enhancement due to G tracts requires A tracts on the same strand.

Base Sequence↗
PubMed1990

Analysis of leaky viral translation termination codons in vivo by transient expression of improved beta-glucuronidase vectors.

Plant RNA viruses commonly exploit leaky translation termination signals in order to express internal protein coding regions. As a first step to elucidate the mechanism(s) by which ribosomes bypass leaky stop codons in vivo, we have devised a system in which readthrough is coupled to the transient expression of beta-glucuronidase (GUS) in tobacco protoplasts. GUS vectors that contain the stop codons and surrounding nucleotides from the readthrough regions of several different RNA viruses were constructed and the plasmids were tested for the ability to direct transient GUS expression. These studies indicated that ribosomes bypass the leaky termination sites at efficiencies ranging from essentially 0 to ca. 5% depending upon the viral sequence. The results suggest that the efficiency of readthrough is determined by the sequence surrounding the stop codon. We describe improved GUS expression vectors and optimized transfection conditions which made it possible to assay low-level translational events.

Base Sequence↗