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W L Gerlach

Publications and source records attributed to W L Gerlach.

At least 19 recordsLinked to original sources
PubMed1992

Extended target-site specificity for a hammerhead ribozyme.

In vitro mutagenesis has been used to systematically mutate the GUC target site cleaved by a synthetic ribozyme based on the catalytic domain of the satellite RNA of tobacco ringspot virus. Amongst the spectrum of changes, it is found that GUC, UUC, CUC, GUA and GUU targets show equivalent rates of cleavage. An AUC target does not cleave, in contrast to observations from other studies. For a GUG target site, the normal ribozyme cannot induce cleavage, but an alteration of the stem-loop in the catalytic domain leads to the formation of a weakly active ribozyme. Certain double mutations, not previously studied, showed slow but discernable cleavage. This mutational approach shows that general rules for cleavage at NUY triplets for the target site of hammerhead ribozymes should be modified. Not all NUY targets cleave under all circumstances, and there are some targets with nucleotides other than U in the centre position which show significant, discernable cleavage.

Autoradiography↗
PubMed1991

Infectious in vitro transcripts from a cloned cDNA of barley yellow dwarf virus.

A full-length cDNA clone of barley yellow dwarf virus (BYDV-PAV serotype) has been constructed and fused to the bacteriophage T7 RNA polymerase promoter. RNA transcripts produced in vitro, either capped or uncapped, were infectious in Triticum monococcum protoplasts. Protoplasts inoculated with in vitro-transcribed BYDV RNA accumulated coat protein, synthesized new viral RNAs, and produced virus particles. Aphid feeding on extracts from protoplasts inoculated with in vitro RNA transcripts can be used to transfer the virus progeny to whole plants. Introduction of mutations which interrupt specific BYDV-PAV open reading frames (ORFs) V and VI eliminated infectivity while an ORF I-mutant remained infectious. Infectious RNA transcripts derived from BYDV cDNA clones will facilitate analysis of the molecular aspects of BYDV infection and further enhance our understanding of this economically important virus.

Animals↗
PubMed1991

A satellite RNA of barley yellow dwarf virus contains a novel hammerhead structure in the self-cleavage domain.

An RNA molecule with properties of a satellite RNA was found in an isolate of barley yellow dwarf virus (BYDV), RPV serotype. It is 322 nucleotide long, single-stranded, and does not hybridize to the viral genome. Dimers of the RNA, which presumably represent replicative intermediates, were able to self-cleave into monomers. In vitro transcripts from cDNA clones were capable of self-cleavage in both the plus (encapsidated) and minus orientations. The sequence flanking the minus strand cleavage site contained a consensus "hammerhead" structure, similar to those found in other self-cleaving satellite RNAs. Although related to the hammerhead structure, sequences flanking the plus strand termini showed differences from the consensus and may be folded into a different structure containing a pseudo-knot.

Base Sequence↗
PubMed1990

Nucleotide sequences of an Australian and a Canadian isolate of potato leafroll luteovirus and their relationships with two European isolates.

The genomes of an Australian and a Canadian isolate of potato leafroll virus have been cloned and sequenced. The sequences of both isolates are similar (about 93%), but the Canadian isolate (PLRV-C) is more closely related (about 98% identity) to a Scottish (PLRV-S) and a Dutch isolate (PLRV-N) than to the Australian isolate (PLRV-A). The 5'-terminal 18 nucleotide residues of PLRV-C, PLRV-A, PLRV-N and beet western yellows virus have 17 residues in common. In contrast, PLRV-S shows no obvious similarity in this region. PLRV-A and PLRV-C genomic sequences have localized regions of marked diversity, in particular a 600 nucleotide residue sequence in the polymerase gene. These data provide a world-wide perspective on the molecular biology of PLRV strains and their comparison with other luteoviruses and related RNA plant viruses suggests that there are two major subgroups in the plant luteoviruses.

Australia↗
PubMed1989

Sequences required for self-catalysed cleavage of the satellite RNA of tobacco ringspot virus.

The satellite RNA of tobacco ringspot virus (sTobRV) undergoes self-catalysed cleavage during replication. A plasmid for in vitro expression of sTobRV has been constructed and used to obtain a library of mutagenized sTobRV sequences. Screening of these mutants has allowed precise definition of the sequences required for (+) and (-) strand cleavage. The sequences and RNA structures associated with cleavage of each strand differ markedly. Cleavage of the (+) strand requires those sequences flanking the site for cleavage to form a 'hammerhead' domain, similar to those found in other satellite and viroid RNA. In contrast, cleavage of the (-) strand requires only a small region of 12 nucleotides (nt) at the site of cleavage, and a sequence of 55 nt positioned elsewhere in the molecule. Comparison with a closely related satellite suggests that a novel RNA structure may be involved in (-) strand cleavage.

Base Sequence↗
PubMed1988

Anaerobically regulated aldolase gene of maize. A chimaeric origin?

The sequence of the anaerobically induced fructose 1,6-bisphosphate aldolase gene of maize is presented. Analysis of the upstream sequences of the aldolase gene reveals a six base-pair sequence (TGGTTT) with perfect homology to one of the sub-regions of the anaerobic regulatory element (ARE) which is responsible for the anaerobic induction of the maize alcohol dehydrogenase 1 gene (Adh1). In the aldolase gene this sequence is located at position -70 relative to the start of transcription, in a small segment proven by functional analysis to be important for expression of the aldolase gene. Since this six base-pair sequence has been shown to be critical for anaerobic induction of the Adh1 mRNA, is in the functional promoter region of aldolase and is also present in a homologous position in Adh2 (another anaerobically-induced gene), we suggest this hexanucleotide is essential for anaerobic regulation of each of these genes. The maize aldolase gene is about 50% homologous at the amino acid level to the animal aldolase gene but has a completely different intron/exon structure. While the rat aldolase gene has nine introns the maize gene has a single large intron near the N terminus of the coding region. Because there is 55% homology downstream from the intron and very little homology upstream, we suggest that the maize gene has acquired a 5' region containing signals for anaerobic regulation and fortuitously adding a new N-terminal region to the protein. We must suppose that the plant gene has lost the remaining introns.

Anaerobiosis↗
PubMed1988

Simple RNA enzymes with new and highly specific endoribonuclease activities.

In vitro mutagenesis of sequences required for the self-catalysed cleavage of a plant virus satellite RNA has allowed definition of an RNA segment with endoribonuclease activity. General rules have been deduced for the design of new RNA enzymes capable of highly specific RNA cleavage, and have been successfully tested against a new target sequence.

Arginine↗
PubMed1988

Sequence and organization of barley yellow dwarf virus genomic RNA.

The nucleotide sequence of the genomic RNA of barley yellow dwarf virus, PAV serotype was determined, except for the 5'-terminal base, and its genome organization deduced. The 5,677 nucleotide genome contains five large open reading frames (ORFs). The genes for the coat protein (1) and the putative viral RNA-dependent RNA polymerase were identified. The latter shows a striking degree of similarity to that of carnation mottle virus (CarMV). By comparison with corona- and retrovirus RNAs, it is proposed that a translational frameshift is involved in expression of the polymerase. An ORF encoding an Mr 49,797 protein (50K ORF) may be translated by in-frame readthrough of the coat protein stop codon. The coat protein, an overlapping 17K ORF, and a 3'6.7K ORF are likely to be expressed via subgenomic mRNAs.

Amino Acid Sequence↗
PubMed1988

The Ds1 transposable element acts as an intron in the mutant allele Adh1-Fm335 and is spliced from the message.

The Ds-induced maize Adh1 allele Adh1-Fm335 retains its anaerobic regulation and normal transcription start site despite the presence of the 405 bp Ds element in the 5' untranslated leader region of the gene. The steady state level of Adh1-specific transcript is reduced to about 1% that of the progenitor or revertant alleles. Run-on transcription studies show that the reduced level of Adh1 specific mRNA is not attributable to a decreased transcription rate. S1 mapping indicates that the Ds element is spliced from the Adh1-Fm335 transcript using a donor site 14 bp into the Ds element and an acceptor site at the 3' junction of the Ds element with the flanking genome DNA.

Alcohol Dehydrogenase↗
PubMed1988

Sequence and identification of the barley yellow dwarf virus coat protein gene.

The nucleotide sequence of the coat protein gene of barley yellow dwarf virus (BYDV, PAV serotype) was determined, and the amino acid sequence was deduced. The open reading frame, encoding a protein of relative molecular mass (Mr) 22,047, was confirmed as the coat protein gene by comparison with amino acid sequences of tryptic peptides derived from dissociated virions. In addition, a fragment of this gene expressed in Escherichia coli produced a product which was recognized by antibodies prepared against purified BYDV virions. An overlapping reading frame encoding an Mr 17,147 protein is contained completely within the coat protein gene.

Amino Acid Sequence↗
PubMed1987

The Ds1 controlling element family in maize and Tripsacum.

Hybridization experiments indicated that the maize genome contains a family of sequences closely related to the Ds1 element originally characterized from the Adh1-Fm335 allele of maize. Examples of these Ds1-related segments were cloned and sequenced. They also had the structural properties of mobile genetic elements, i.e., similar length and internal sequence homology with Ds1, 10- or 11-bp terminal inverted repeats, and characteristic duplications of flanking genomic DNA. All sequences with 11-bp terminal inverted repeats were flanked by 8-bp duplications, but the duplication flanking one sequence with 10-bp inverted repeats was only 6 bp. Similar Ds1-related sequences were cloned from Tripsacum dactyloides. They showed no more divergence from the maize sequences than the individual maize sequences showed when compared with each other. No consensus sequence was evident for the sites at which these sequences had inserted in genomic DNA.

Base Sequence↗
PubMed1985

Molecular analysis of the alcohol dehydrogenase 2 (Adh2) gene of maize.

The Adh2 gene of maize has a nucleotide sequence closely related to that of the maize Adh1 gene indicating that the two genes arose from a progenitor gene by a duplication event. The coding regions are 82% conserved at the nucleotide level and 87% conserved at the amino acid level. Each gene has nine introns in identical positions but their nucleotide sequences and lengths differ. Adh2 encodes a single mRNA and has a possible polyadenylation signal, AATAAT, fifteen bases upstream from the polyadenylation site. The Adh2 gene together with the Adh1 gene is induced by anaerobic conditions and under these conditions produces an increased level of mRNA. The 5' untranslated regions of the transcripts of Adh1 (100 bp) and Adh2 (126 bp) have diverged in sequence except for a conserved region which may be important for their anaerobic-specific translation. The 3' and 5' flanking sequences of the two genes are also divergent except for 11 bp of precise homology around the TATA boxes and three other 8 bp sequences further upstream. One of the common 8 bp sequences (CACCTCCC) is in a similar position (-150 bp to -200 bp) in the two genes and may have a regulatory function in gene expression.

Alcohol Dehydrogenase↗
PubMed1979

Cloning and characterization of ribosomal RNA genes from wheat and barley.

Wheat and barley DNA enriched for ribosomal RNA genes was isolated from actinomycin D-CsCl gradients and used to clone the ribosomal repeating units in the plasmid pAC184. All five chimeric plasmids isolated which contained wheat rDNA and eleven of the thirteen which had barley rDNA were stable and included full length ribosomal repeating units. Physical maps of all length variants cloned have been constructed using the restriction endonucleases Eco Rl, Bam Hl, Bgl II, Hind III and Sal I. Length variation in the repeat units was attributed to differences in the spacer regions. Comparison of Hae III and Hpa II digestion of cereal rDNAs and the cloned repeats suggests that most methylated cytosines in natural rDNA are in -CpG-. Incomplete methylation occurs at specific Bam Hl sites in barley DNA. Detectable quantities of ribosomal spacer sequences are not present at any genomic locations other than those of the ribosomal RNA gene repeats.

Base Sequence↗