Biomedical subjects

R A Wing

Publications and source records attributed to R A Wing.

41 records · Page 3Linked to original sources

PubMed1991

Isolation of molecular markers from specific chromosomal intervals using DNA pools from existing mapping populations.

We present a general method for isolating molecular markers specific to any region of a chromosome using existing mapping populations. Two pools of DNA from individuals homozygous for opposing alleles for a targeted chromosomal interval, defined by two or more linked RFLP markers, are constructed from members of an existing mapping population. The DNA pools are then screened for polymorphism using random oligonucleotide primers and PCR (1). Polymorphic DNA bands should represent DNA sequences within or adjacent to the selected interval. We tested this method in tomato using two genomic intervals containing genes responsible for regulating pedicle abscission (jointless) and fruit ripening (non-ripening). DNA pools containing 7 to 14 F2 individuals for each interval were screened with 200 random primers. Three polymorphic markers were thus identified, two of which were subsequently shown to be tightly linked to the selected intervals. The third marker mapped to the same chromosome (11) but 45 cM away from the selected interval. A particularly attractive attribute of this method is that a single mapping population can be used to target any interval in the genome. Although this method has been demonstrated in tomato, it should be applicable to any sexually reproducing organism for which segregating populations are being used to construct genetic linkage maps.

Base Sequence↗

PubMed1991

Promoter analysis of genes that are coordinately expressed during pollen development reveals pollen-specific enhancer sequences and shared regulatory elements.

We have investigated the functional organization and properties of cis regulatory elements in the promoter regions of two genes from tomato (LAT52 and LAT59) that are preferentially and coordinately expressed during pollen maturation. Promoter deletion analysis in transgenic plants demonstrated that only minimal (less than 200 bp) promoter proximal regions are required for developmentally regulated expression in pollen and in specific cell types of the sporophyte. Cis-acting regulatory regions of these two promoters and of a third pollen-expressed promoter (LAT56) were characterized in detail using a transient expression assay. We identified two upstream activator regions in the LAT52 promoter and further showed that a 19-bp segment from one of those regions enhanced expression of the heterologous CaMV35S promoter in pollen. Similarities in sequence between crucial cis elements provide evidence that shared regulatory elements are involved in the coordinate expression of the LAT genes during microsporogenesis.

Base Sequence↗

PubMed1990

Molecular and genetic characterization of two pollen-expressed genes that have sequence similarity to pectate lyases of the plant pathogen Erwinia.

A set of cDNAs that are expressed in tomato anthers were isolated. We further characterized two of these cDNAs (LAT56 and LAT59) and their corresponding genomic clones. LAT56 and LAT59 show low levels of steady-state mRNA in immature anthers and maximal levels in mature anthers and pollen. The LAT56 and LAT59 genes are single-copy in the tomato genome, and are linked on chromosome 3, approximately 5 cM apart. Although these cDNAs did not cross-hybridize, their deduced protein sequences (P56 and P59) have 54% amino acid identity. The LAT56 and LAT59 genes each have two introns, but they are located in non-homologous positions. P56 and P59 show significant protein sequence similarity to pectate lyases of plant pathogenic bacteria. The similarity of P56 and P59 to the bacterial pectate lyases is equivalent to the homology described for different pectate lyase sequences of the genus Erwinia. We suggest that the pollen expression of LAT56 and LAT59 might relate to a requirement for pectin degradation during pollen tube growth.

Amino Acid Sequence↗

PubMed1989

The chloroplast genome exists in multimeric forms.

Chloroplast DNA conformation was analyzed by pulse-field gel electrophoresis. We found that spinach leaf chloroplast DNA molecules exist in at least four distinct forms with the apparent molecular weights of monomer, dimer, trimer, and tetramer. Two-dimensional gel analysis of DNA after UV nicking and in the presence of ethidium bromide indicates that they are not isomers that differ in superhelical density. DNA gyrase decatenation analysis demonstrates that the majority of the DNA molecules are oligomers rather than catenanes. The relative amounts of monomer, dimer, trimer, and tetramer forms, quantitated by molecular hybridization, are 1, 1/3, 1/9, and 1/27, respectively, and do not change during leaf maturation. The possible mechanisms of chloroplast DNA oligomer formation are discussed.

Journal Article↗

PubMed1988

Intracellular precursors and secretion of alkaline extracellular protease of Yarrowia lipolytica.

Processing and secretion of the alkaline extracellular protease (AEP) from the yeast Yarrowia lipolytica was studied by pulse-chase and immunoprecipitation experiments. Over half of newly synthesized AEP was secreted by 6 min. Over 99% of AEP activity which was external to the cytoplasmic membrane was located in the supernatant medium. Polypeptides of 55, 52, 44, 36, and 32 kilodaltons (55K, 52K, 44K, 36K, and 32K polypeptides) were immunoprecipitated from [3H]leucine-labeled cell extracts by rabbit antibodies raised against mature, secreted AEP (32K polypeptide). Experiments with tunicamycin and endoglycosidase H indicated that the 55K, 52K, and 44K polypeptides contained about 2 kilodaltons of N-linked oligosaccharide and that the 36K and 32K polypeptides contained none. Results of pulse-chase experiments did not fit a simple precursor-product relationship of 55K----52K----44K----36K----32K. In fact, maximum labeling intensity of the 52K polypeptide occurred later than for the 44K and 36K polypeptides. Secretion of polypeptides of 19 and 20 kilodaltons derived from the proregion of AEP indicated that one major processing pathway was 55K----52K----32K. The gene coding for AEP (XPR2) was cloned and sequenced. The sequence and the immunoprecipitation results suggest that AEP is originally synthesized with an additional preproI-proII-proIII amino-terminal region. Processing definitely involves cleavage(s) after pairs of basic amino acids and the addition of one N-linked oligosaccharide. Signal peptidase cleavage, dipeptidyl aminopeptidase cleavages, and at least one additional proteolytic cleavage may also be involved.

Amino Acid Sequence↗