A high efficient approach used for BAC-contig extension of Oryza sativa with PCR screening the BAC clone pools.

To extend 8 BAC contigs, which were previously located in the 56.1-68 cM region of the chromosome 4 of the Oryza sativa indica GuangLuAi4, 14 pairs of primers were designed according to the terminal sequences of the existing seed BACs and were deliberately divided into 3 groups. With the 3 groups of primer mixtures, 233 pools of BAC DNA that represent 22 368 BAC clones from O.sativa indica GuangLuAi4 genomic library were screened. 65 positive clones corresponding to the 8 contigs were isolated and 29 clones of them were confirmed to be extended to the seed BACs by end-sequencing and fingerprinting. The protocol greatly enhanced the efficiency of the contig extension and was also superior for its specificity, sensitivity and reusability to the colony in situ hybridization which is a conventional method employed in contig extension and physical map construction.

A HU-like protein binds to specific sites within nod promoters of Rhizobium leguminosarum.

Nodulation genes (nod) of rhizobia are essential for establishment of its symbiosis with specific legume hosts and are usually located on the Sym(biosis) megaplasmid. In this work we identified a new Sym plasmid independent protein in Rhizobium leguminosarum, Px, by its ability to bind to nod promoters and induce DNA bending. Depending upon its concentrations relative to DNA templates, Px could either stimulate or inhibit in vitro transcription of the major regulatory nodulation gene nodD. This may result from its property to bind to specific sites within nod promoters at lower concentration or in the presence of competitor calf thymus DNA but nonspecifically associate with DNA at higher levels or in the absence of competitors. Its binding sites within nodD and nodF promoters were determined by DNase I footprinting but showed no sequence consensus. N-terminal sequencing and Western blot revealed that Px belongs to the HU class of prokaryotic histone-like proteins. Its binding feature and functioning mechanism were discussed in the light of this discovery.

A region of a Sym plasmid of Rhizobium leguminosarum biovar phaseoli has similarity to prokaryotic insertion sequences and to eukaryotic integrases.

Near the nod and nif genes of the Sym plasmid pRP2JI of Rhizobium leguminosarum biovar phaseoli are three open reading frames whose deduced polypeptide products have similarities to those of genes in bacterial insertion sequences. The similarity of one of these ORFs was significantly greater to that of the integrase region of pol proteins of eukaryotic retroviruses and transposable elements in animals and plants than it was to the transposases of prokaryotic insertion sequences. In the noncoding region of the IS-like element, there was a sequence similar to that which had been identified close to nod genes in Azorhizobium caulinodans.

Sequence and regulation of psrA, a gene on the Sym plasmid of Rhizobium leguminosarum biover phaseoli which inhibits transcription of the psi genes.

The psr region of Rhizobium leguminosarum biovar phaseoli had originally been recognized on the basis of its ability to repress the transcription of the psi genes, one of which, psiA, inhibits exopolysaccharide synthesis when cloned in multi-copy plasmids. Both psr and psi are located on the symbiotic plasmid pRP2JI. The psrA gene was localized and sequenced. The deduced amino acid sequence of PsrA was shown to have similarity to the DNA-binding region of a family of other transcriptional regulators, consistent with its known effects on the expression of psi. The transcription of psrA itself appears to be constitutive in free-living Rhizobium, but is regulated by another gene on the Sym plasmid pRP2JI.

Secretion of the Rhizobium leguminosarum nodulation protein NodO by haemolysin-type systems.

The Rhizobium leguminosarum biovar viciae nodulation protein NodO is partially homologous to haemolysin of Escherichia coli and, like haemolysin, is secreted into the growth medium. The NodO protein can be secreted by a strain of E. coli carrying the cloned nodO gene plus the haemolysin secretion genes hlyBD, in a process that also requires the outer membrane protein encoded by tolC. The related protease secretion genes, prtDEF, from Erwinia chrysanthemi also enable E. coli to secrete NodO. The Rhizobium genes encoding the proteins required for NodO secretion are unlinked to nodO and are unlike other nod genes, since they do not require flavonoids or NodO for their expression. Although proteins similar to NodO were not found in rhizobia other than R. leguminosarum bv. viciae, several rhizobia and an Agrobacterium strain containing the cloned nodO gene were found to have the ability to secrete NodO. These observations indicate that a wide range of the Rhizobiaceae have a protein secretion mechanism analogous to that which secretes haemolysin and related toxins and proteases in the ENterobacteriaceae.

Sequencing with the large fragment of DNA polymerase I from Bacillus stearothermophilus.

The large fragment of DNA polymerase I, isolated from Bacillus stearothermophilus, was used for dideoxy sequencing. This heat-stable enzyme permits performing sequencing reactions at high temperature to melt secondary structure and results in uniform band intensities and low background on the autoradiogram. The enzyme can be used in the standard Sanger one-step protocol or in a two-step protocol which separates the labeling reaction from the elongation-termination reaction. The enzyme can be used in double-stranded sequencing. 35S-labeled nucleotides may be used instead of 32P-labeled nucleotides. Both 7-deaza-dGTP and dITP can be used during the reaction in order to minimize band compression on the gel. Results presented here indicate that this enzyme should be a useful tool for sequence determination.

Rapid method for identifying subclones bridging gaps between contiguous DNA sequences.

Randomly sequencing of a limited number of unidirectional deletion subclones efficiently generated contiguous DNA sequences with some gaps between them. Gap-bridging subclones were identified by agarose gel electrophoresis. Those located at either end of each individual contiguous sequence were used as standard marker molecules; the subclones between them proved to be correctly identified in most cases. A 4064 bp DNA fragment containing the psr gene was sequenced with the strategy described in this paper.

Evidence that DNA involved in the expression of nodulation (nod) genes in Rhizobium binds to the product of the regulatory gene nodD.

In Rhizobium leguminosarum biovar viciae, the regulatory nodulation nodD gene has at least two functions. It constitutively represses its own transcription and in the presence of inducer flavonoid molecules, it activates the expression of two other nod gene transcriptional units, nodABCIJ and nodFE. Upstream of nodA and nodF is a conserved sequence, the nod box, which has been implicated in nodD-mediated transcriptional activation of these genes. DNA fragments spanning the nod boxes that precede nodA and nodF were end-labelled and were exposed to cell-free extracts obtained from strains of Rhizobium. Using the gel retardation technique, it was shown that a complex between protein and these DNA fragments was formed, but only if the extract contained a functional nodD gene. Evidence that the protein that binds to the regulatory sequences is the nodD gene product came from the observation that a complex was formed between the nod box preceding nodA and protein from a cell-free extract isolated from Escherichia coli containing the cloned nodD gene. Extracts from Rhizobium strains containing mutant forms of nodD which were specifically affected in autoregulation or in flavonoid-dependent activation formed either no protein DNA complex or formed a complex with altered mobility compared to that obtained with extracts from wild-type strains.

Heat-stable DNA polymerase I large fragment resolves hairpin structure in DNA sequencing.

A heat-stable large fragment was obtained by subtilisin digestion of DNA polymerase, prepared from Bacillus stearothermophilus. The dideoxy sequencing method, combined with the use of M13 vector has proved to be the most powerful one for obtaining the sequences of large genomes. However, the hairpin structure formed along the single-stranded DNA template often prevents the DNA polymerase from moving on, with the result that no sequence information can be obtained. The heat-stable large fragment that we have obtained has proved to be the most active at 65 degrees C. When the sequencing reaction was carried out at this temperature, the hairpin structure was resolved and the sequencing gels obtained were satisfactory.

Buffer gradient gels and 35S label as an aid to rapid DNA sequence determination.

Two methods for increasing the length of DNA sequence data that can be read off a polyacrylamide gel are described. We have developed a rapid way to pour a buffer concentration gradient gel that, by altering the vertical band separation on an autoradiograph, allows more sequence to be obtained from a gel. We also show that the use of deoxyadenosine 5'-(alpha-[35S]thio)triphosphate as the label incorporated in dideoxynucleotide sequence reactions increases the sharpness of the bands on an autoradiograph and so increases the resolution achieved.

Sequencing of large double-stranded DNA using the dideoxy sequencing technique.

The dideoxy sequencing technique has been applied to the direct sequencing of large double-stranded DNA molecules with a small single-stranded primer. For instance, the method was applied to the lambda genome, which contains 48 502 base-pairs (Sanger F, Coulson AR, Hong GF, Hill D & Petersen GB, 1982, J. Mol. Biol., in press), and the coding region for gene W identified. The procedure proves useful in the sequence analysis of a large number of different mutations in a particular region and in the analysis of eukaryotic DNA cloned in plasmids, phages, and cosmids.

Rapid synthesis of oligodeoxyribonucleotides VI. Efficient, mechanised synthesis of heptadecadeoxyribonucleotides by an improved solid phase phosphotriester route.

Efficient mechanised synthesis of heptadecadeoxyribonucleotides has been achieved on an economically small scale by an improved solid phase phosphotriester method on a polydimethylacrylamide resin. Improvements were made in the preparation of dinucleotide building blocks, reaction conditions for oligonucleotide assembly and in purification of deprotected oligonucleotides by h.p.l.c. Several milligrams of pure heptadecamers were obtained. Two of the heptadecamers were designed for sequencing in opposite directions of DNA cloned in phage M13mp2.

A method for sequencing single-stranded cloned DNA in both directions.

A DNA sequencing method has been developed whereby DNA that has been cloned in a single-stranded bacteriophage vector can be sequenced from both ends. The method involves first making a minus-strand sense template from a single-stranded insert in the vector M13mp2 using a flanking primer, and then sequencing the synthesized template using the dideoxynucleotide termination method (Sanger et al., 1977, 1980) with a second primer. Special conditions are described under which the first primer is easily removed after making the template, and sequencing in the opposite direction can be done in the normal way (Sanger et al., 1980) without separating the double strands. This method renders it possible to read up to twice the amount of sequence data from a long insert and also to check short inserts by producing complementary sequence patterns.