Inconsistencies between human genetic cytolocations and those derived using genomic sequence.

One result of the publishing of the human genome sequence is the ability to define objects through their position on the consensus sequence. While this has simplified the process of creating order maps for genes on a chromosome, it has created discrepancies between the published cytolocations of human genes, as presented through genetic references, and those locations derived computationally from the genomic sequence. For the 6,830 records with HUGO gene symbols shared between the online version of Mendelian Inheritance in Man and Ensembl, 18% of the records have a discrepancy of at least one cytogenetic band between the datasets. Discordance between data sets at this frequency would have a significant impact on the utility of datasets created by the amalgamation of numerous biological databases.

Soybean DapA mutations encoding lysine-insensitive dihydrodipicolinate synthase.

In plants, the rate-limiting step in the pathway for lysine synthesis is catalyzed by the enzyme dihydrodipicolinate synthase (DS), which is encoded by the DapA gene. We previously cloned the soybean (Glycine max cv. Century) DapA gene in Escherichia coli to express functional soybean DS protein. Like the wild-type soybean DS enzyme, the DS activity encoded by the cloned gene was extremely sensitive to feedback inhibition by micromolar concentrations of lysine. Three mutants of the soybean DapA gene were constructed using PCR: one with a single amino acid substitution at codon 104, another with a single amino acid substitution at codon 112, and a mutant containing both modifications. When expressed in E. coli, the mutant DS activities were insensitive to lysine at concentrations up to 1 mM.

Cloning and expression of the soybean DapA gene encoding dihydrodipicolinate synthase.

The rate-limiting step in the pathway for lysine synthesis in plants is catalyzed by the enzyme dihydrodipicolinate synthase (DS). We have cloned the portion of the soybean (Glycine max cv. Century) DapA cDNA that encodes the mature DS protein. Expression of the cloned soybean cDNA, as a lacZ fusion protein was selected in a dapA- Escherichia coli auxotroph. The DS activity of the fusion protein was characterized in E. coli extracts. The DS activity of the fusion protein was inhibited by lysine concentrations that also inhibited native soybean DS, while E. coli DS activity was much less sensitive to inhibition by lysine.

Posttranscriptional accumulation of chloroplast tufA (elongation factor gene) mRNA during chloroplast development in Chlamydomonas reinhardtii.

Light induces chloroplast (Cp) differentiation in dark-grown y-1 strains of Chlamydomonas reinhardtii. Slot blot analysis was used to quantitate tufA, psbA, psbK, rbcL, and 16S rRNA transcript accumulation and transcription during Cp differentiation. When etiolated cc-125 y-1 cells were illuminated for 5 h, a 1710 bp tufA mRNA accumulated up to 5-fold while the psbA, rbcL, and 16S rRNA transcripts accumulated less than 1.5-fold. The tufA gene encodes translational elongation factor EF-Tu. The light-induced accumulation of tufA mRNA did not occur in cc-1931, a strain that does not become etiolated in darkness. Pulse labelling was used to measure the transcription of Cp transcripts during tufA mRNA accumulation, and no detectable change in tufA transcription was observed. These results imply that the half life of the tufA transcript increases during the greening process.

3' transcript mapping of the Chlamydomonas reinhardtii chloroplast psbK mRNA using a novel method to prepare 3' end-labeled single-stranded DNA probes.

We have developed a novel method for the rapid preparation of large quantities of 3' end-labeled single-stranded (ss) DNA (ssDNA) probes for transcript mapping. A recombinant phagemid vector containing the probe sequence was used to raise large quantities of ssDNA. Based on the DNA sequence of the probe, an oligonucleotide primer complementary to the ssDNA probe was synthesized. Annealing of the primer to the purified ssDNA phagemid clone produced a short double-stranded DNA duplex containing a restriction site, which was then cut with a restriction enzyme to generate a 5' overhang of the primer strand. The phagemid DNA was labeled at the 3' end with the Klenow fragment of polymerase I. The low melting temperatures of the short primer-phagemid duplex caused the primer to dissociate during the hybridization of the probe to algal RNA. For this reason, the probe can be used for S1 mapping without further purification. This method was used to map the 3' end of the Chlamydomonas reinhardtii chloroplast psbK transcript.

Darkness and antibiotics increase the steady-state transcripts of the elongation factor gene (tuf) in Chlamydomonas reinhardtii.

A plasmid library of chloroplast (Cp) DNA from Chlamydomonas reinhardtii was used to screen for transcripts which respond to light. A transcript of R03, a 1,300 bp EcoRI fragment, was identified as a message which accumulates in darkness. The transcribed region of R03 showed extensive sequence homology with the Escherichia coli elongation factor gene, tufA. A gene-specific probe was constructed. Northern blots were used to study the extent and kinetics of accumulation of this transcript in darkness and in the presence of antibiotic inhibitors of Cp ribosomes. For comparison, the effects of darkness and antibiotics on the steady state levels of psbA, rbcL, and 16S rRNA were also studied. We conclude that the tuf transcript shows the greatest increase in darkness and in the presence of antibiotics.