CYTOGENETICAL TREATISE OF INDIAN REPRESENTATIVE SPECIES OF CUCUMIS. A KARYOTYPIC APPROACH.

Karyomorphological studies have been carried out in nine species and five varieties of the genus Cucumis representing Indian gene pool. The present investigations reveal the occurrence of two somatic chromosome numbers 2n = 14, 24 in the genus. C. ritchiei and C. indicus the two new species, were found to be having somatic chromosome numbers of 2n = 24 and 2n = 20 respectively. The wild species viz. C. hystrix, C. setosus, C. prophetarum, C. dipsaceus, C. indicus have very less number of median-centromeric chromosomes, high asymmetry indices, while melon groups have intermediate number of median -centromeric chromosomes. C. sativus, C. callosus, C. ritchiei show lesser number median-cen-tromeric chromosomes and very less asymmetry indices. The importance of karyotypic variation with respect to speciation within the genus Cucumis have been discussed.

Efficacy of random primer-pair arrays in plant genome analysis: a case study of Cucumis (Cucurbitaceae) for identification of wild and cultivated species.

The efficacy of random primer-pair arrays compared to conventional RAPD method with a single decamer primer was evaluated using DNA from two species of Cucumis. The banding patterns of amplicons revealed enhanced utility of primer-pair arrays over conventional RAPDs, producing more bands and a higher degree of polymorphism, both at intra- and inter-specific levels. Amplification produced by both methods clearly distinguished a wild from a cultivated species of the genus Cucumis. The main advantage of the primer-pair RAPD over single-primer-based RAPD is the increase in the number of reactions and amplification products in the form of novel/unique bands with a limited number of primers. It also enables the generation of reliable amplicons with a large number of polymorphic bands, which can be linked to gene-governing traits, allowing sequence-characterized partial genome analysis.

An N-terminally truncated RpoS (sigma(S)) protein in Escherichia coli is active in vivo and exhibits normal environmental regulation even in the absence of rpoS transcriptional and translational control signals.

RpoS (sigma(S)) in Escherichia coli is a stationary-phase-specific primary sigma factor of RNA polymerase which is 330 amino acids long and belongs to the eubacterial sigma(70) family of proteins. Conserved domain 1.1 at the N-terminal end of sigma(70) has been shown to be essential for RNA polymerase function, and its deletion has been shown to result in a dominant-lethal phenotype. We now report that a sigma(S) variant with a deletion of its N-terminal 50 amino acids (sigma(S)Delta1-50), when expressed in vivo either from a chromosomal rpoS::IS10 allele (in rho mutant strains) or from a plasmid-borne arabinose-inducible promoter, is as proficient as the wild type in directing transcription from the proU P1 promoter; at three other sigma(S)-dependent promoters that were tested (osmY, katE, and csiD), the truncated protein exhibited a three- to sevenfold reduced range of activities. Catabolite repression at the csiD promoter (which requires both sigma(S) and cyclic AMP [cAMP]-cAMP receptor protein for its activity) was also preserved in the strain expressing sigma(S)Delta1-50. The intracellular content of sigma(S)Delta1-50 was regulated by culture variables such as growth phase, osmolarity, and temperature in the same manner as that described earlier for sigma(S), even when the truncated protein was expressed from a template that possessed neither the transcriptional nor the translational control elements of wild-type rpoS. Our results indicate that, unlike that in sigma(70), the N-terminal domain in sigma(S) may not be essential for the protein to function as a sigma factor in vivo. Furthermore, our results suggest that the induction of sigma(S)-specific promoters in stationary phase and during growth under conditions of high osmolarity or low temperature is mediated primarily through the regulation of sigma(S) protein degradation.

In vivo expression from the RpoS-dependent P1 promoter of the osmotically regulated proU operon in Escherichia coli and Salmonella enterica serovar Typhimurium: activation by rho and hns mutations and by cold stress.

Unlike the sigma(70)-controlled P2 promoter for the osmotically regulated proU operon of Escherichia coli and Salmonella enterica serovar Typhimurium, the sigma(s)-controlled P1 promoter situated further upstream appears not to contribute to expression of the proU structural genes under ordinary growth conditions. For S. enterica proU P1, there is evidence that promoter crypticity is the result of a transcription attenuation phenomenon which is relieved by the deletion of a 22-base C-rich segment in the transcript. In this study, we have sought to identify growth conditions and trans-acting mutations which activate in vivo expression from proU P1. The cryptic S. enterica proU P1 promoter was activated, individually and additively, in a rho mutant (which is defective in the transcription termination factor Rho) as well as by growth at 10 degrees C. The E. coli proU P1 promoter was also cryptic in constructs that carried 1.2 kb of downstream proU sequence, and in these cases activation of in vivo expression was achieved either by a rho mutation during growth at 10 degrees C or by an hns null mutation (affecting the nucleoid protein H-NS) at 30 degrees C. The rho mutation had no effect at either 10 or 30 degrees C on in vivo expression from two other sigma(s)-controlled promoters tested, those for osmY and csiD. In cells lacking the RNA-binding regulator protein Hfq, induction of E. coli proU P1 at 10 degrees C and by hns mutation at 30 degrees C was still observed, although the hfq mutation was associated with a reduction in the absolute levels of P1 expression. Our results suggest that expression from proU P1 is modulated both by nucleoid structure and by Rho-mediated transcription attenuation and that this promoter may be physiologically important for proU operon expression during low-temperature growth.

Evidence for transcription attenuation rendering cryptic a sigmaS-dependent promoter of the osmotically regulated proU operon of Salmonella typhimurium.

The osmotically regulated proU locus in Escherichia coli has two promoters, P1 and P2, that are recognized, respectively, by the sigmaS- and sigma70-bearing RNA polymerase holoenzymes. However, the equivalent of the P1 promoter does not appear to exist in Salmonella typhimurium. We demonstrate in this study that wild-type S. typhimurium has a cryptic P1 promoter that is recognized by sigmaS RNA polymerase in vitro and that a 22-bp deletion from +63 to +84 (relative to the start site of transcription) confers sigmaS-dependent in vivo expression of a reporter gene fusion to P1. Primer extension analysis of RNA isolated from cells carrying the wild-type and mutant S. typhimurium proU constructs indicated that a primer which hybridizes proximal to +60 is able to detect P1-initiated transcripts from both constructs but a primer which hybridizes distal to +85 is able to do so only from the latter. Our results suggest that the sigmaS-controlled proU P1 promoter in S. typhimurium may be rendered cryptic because of factor-dependent transcription attenuation within a short distance downstream of the promoter start site.

Effects of H-NS and potassium glutamate on sigmaS- and sigma70-directed transcription in vitro from osmotically regulated P1 and P2 promoters of proU in Escherichia coli.

We have used supercoiled DNA templates in this study to demonstrate that transcription in vitro from the P1 and P2 promoters of the osmoresponsive proU operon of Escherichia coli is preferentially mediated by the sigma(s) and sigma70-bearing RNA polymerase holoenzymes, respectively. Addition of potassium glutamate resulted in the activation of transcription from both P1 and P2 and also led to a pronounced enhancement of sigma(s) selectivity at the P1 promoter. Transcription from P2, and to a lesser extent from P1, was inhibited by the nucleoid protein H-NS but only in the absence of potassium glutamate. This study validates the existence of dual promoters with dual specificities for proU transcription. Our results also support the proposals that potassium, which is known to accumulate in cells grown at high osmolarity, is at least partially responsible for effecting the in vivo induction of proU transcription and that it does so through two mechanisms, directly by the activation of RNA polymerase and indirectly by the relief of repression imposed by H-NS.

Multiple mechanisms contribute to osmotic inducibility of proU operon expression in Escherichia coli: demonstration of two osmoresponsive promoters and of a negative regulatory element within the first structural gene.

Transcription of the proU operon in Escherichia coli is induced several hundredfold upon growth of cells in media of elevated osmolarity. A low-copy-number promoter-cloning plasmid vector, with lacZ as the reporter gene, was used for assaying the osmoresponsive promoter activity of each of various lengths of proU DNA, generated by cloning of discrete restriction fragments and by an exonuclease III-mediated deletion approach. The results indicate that expression of proU in E. coli is directed from two promoters, one (P2) characterized earlier by other workers with the start site of transcription 60 nucleotides upstream of the initiation codon of the first structural gene (proV), and the other (P1) situated 250 nucleotides upstream of proV. Furthermore, a region of DNA within proV was shown to be involved in negative regulation of proU transcription; phage Mu dII1681-generated lac fusions in the early region of proV also exhibited partial derepression of proU regulation, in comparison with fusions further downstream in the operon. Sequences around promoter P1, sequences around P2, and the promoter-downstream negative regulatory element, respectively, conferred approximately 5-, 8-, and 25-fold osmoresponsivity on proU expression. Within the region genetically defined to encode the negative regulatory element, there is a 116-nucleotide stretch that is absolutely conserved between the proU operons of E. coli and Salmonella typhimurium and has the capability of exhibiting alternative secondary structure. Insertion of this region of DNA into each of two different plasmid vectors was associated with a marked reduction in the mean topological linking number in plasmid molecules isolated from cultures grown in high-osmolarity medium. We propose that this region of DNA undergoes reversible transition to an underwound DNA conformation under high-osmolarity growth conditions and that this transition mediates its regulatory effect on proU expression.

Molecular cloning of an osmoregulatory locus in Escherichia coli: increased proU gene dosage results in enhanced osmotolerance.

The proU locus in Escherichia coli encodes an important osmoregulatory function which mediates the growth-promoting effect of L-proline and glycine betaine in high-osmolarity media. This locus was cloned, in contiguity with a closely linked Tn10 insertion, onto a multicopy plasmid directly from the E. coli chromosome. For a given level of osmotic stress, the magnitude of osmoresponsive induction of a single-copy proU::lac fusion was reduced in strains with multiple copies of the proU+ genes; in comparison with haploid proU+ strains, strains with the multicopy proU+ plasmids also exhibited enhanced osmotolerance in media supplemented with 1 mM L-proline or glycine betaine. Experiments involving subcloning, Tn1000 mutagenesis, and interplasmid complementation in a deletion mutant provided evidence for the presence at this locus of two cistrons, both of which are necessary for the expression of ProU function. We propose the designations proU for the gene originally identified by the proU224::Mu d1(lac Ap) insertion and proV for the gene upstream (that is, counterclockwise) of proU.