We investigated the viability of Escherichia coli cells during long-term cultivation in Brain Heart Infusion (BHI) medium and observed that the number of viable cells increased, then decreased, and increased again, in this medium, and finally the cells died out within about 10 days. This cell death may result from an increase in the pH of the medium. After repeated cultivation in BHI, bacterial cells that did not die out even under conditions of further cultivation were obtainable from cultures showing a stabilized viable count. We propose that long-term cultivation in BHI medium is a good system for studying growth phase-specific events in E. coli cells, because the total life-cycle of a population of E. coli, including exponential growth, stationary phase, and extinction, can be seen during a period of only about 10 days. Also, this system clearly allows detection of a phenotype that may not be detectable in orther commonly used media. Moreover, in this report, we show that mutants displaying the GASP (growth advantage in stationary phase) phenotype appear at high frequency under long-term cultivation conditions.
The 5' flanking and coding regions of the psbA gene were sequenced to clarify relationships among several taxa discovered recently in thalloid liverwort Conocephalum conicum. Twenty six samples included in this study represent five “cryptic species” detected in worldwide collection, five groups discovered from Japan and finally three “chemo-types” with different dominant volatile component. Pairwise differences in nucleotide sequences of coding region between samples of Conocephalum conicum varied greatly depending on the combination, ranging from 0.001 to 0.018 per site. In total, seven amino acid substitutions were found among all samples. NJ and parsimony trees among the taxa were constructed for the first time. The phylogenetic relationships among taxa were basically consistent with those inferred from isozyme and morphological studies. Conocephalum conicumspecies FS and T taxon found in Japan have identical sequence of psbA gene, suggesting that they are conspecific. Similarly, YFS and KYT taxa cluster together with FS cryptic species, and “chemo-types” I and II with “cryptic species” J. A chemotype III was very different from any other taxa of C. conicum. These results suggest that morphological species, Conocephalum conicum is highly differentiated at the molecular level and some of the taxa may in fact represents different species as previous studies suggested.
In the sexual reproduction of Paramecium tetraurelia, the somatic nucleus (macronucleus) undergoes massive genomic rearrangement, including gene amplification and excision of internal eliminated sequences (IESs), in its normal developmental process. Strain d4-662, one of the pawn mutants, is a behavioral mutant of P. tetraurelia that carries a recessive allele of pwB662. The pwB gene in the macronucleus of the strain has an insertion of the IES because a base substitution within the IES prevents its excision during gene rearrangement. Cultures of this strain frequently contain cells reverting to the wild type in the behavioral phenotype. The mutant and revertant cells maintained stable clonal phenotypes under the various environmental conditions examined unless they underwent sexual reproduction. After sexual reproduction, both mutant and revertant produced 2.7-7.1% reverted progeny. A molecular analysis performed on the macronuclear DNA of the mutant and revertant of d4-662 showed that much less than 1% of the mutant IES was precisely excised at every sexual reproduction of the strain. Therefore, the alternative phenotype of strain d4-662 seems to be caused by an alternative excision of the mutant IES.
Homoeoalleles of Ncc confer nucleus-cytoplasm (NC) compatibility on NC hybrids of wheat with the D plasmon of Aegilops squarrosa. To dissect the chromosomal region containing Ncc, a RAPD marker linked to the Ncc-tmp1A locus, which is located on chromosome 1A of T. timopheevi, was sequenced and converted to a PCR-based sequence-tagged-site (STS) marker. Five single nucleotide polymorphisms (SNPs) between T. timopheevi and T. turgidum. were detected in a 509-bp genomic DNA fragment. Based on the SNPs, the STS alleles in 164 accessions from emmer wheat, timopheevi wheat and two einkorn wheats, T. urartu and T. boeoticum were surveyed by PCR-RFLP analysis. The sequence comparisons and PCR-RFLP analyses revealed nine alleles based on six SNPs. These SNPs were highly conserved within each group of wheat, and all groups could be distinguished by particular combinations of the SNPs. All accessions of T. urartu had one unique STS allele as compared with the others. Our results indicate that the SNPs in the STS marker linked to the Ncc-tmp1A locus would be informative for studies of the differentiation of chromosome 1A in wheat.
Aegilops caudata L. is an annual wild relative of wheat distributed over the northeastern Mediterranean basin. It consists of two taxonomic varieties, var. typica with awnless lateral spikelets and var. polyathera with awned lateral spikelets. To clarify the variation and the geographical distribution of the genotypes controlling the diagnostic spike morphology of the two taxonomic varieties, three crossing experiments were carried out. First, two varieties collected from nine sympatric populations in the Aegean islands were crossed reciprocally. All of the F1 hybrids were var. typica and the segregation ratio in the F2 generation was 3 typica: 1 polyathera. Secondly, 13 typica accessions collected from the entire distribution area of the variety were crossed with a common polyathera accession. The F1 hybrids involving eight typica accessions from Greece and West Anatolia were var. typica, while those involving five typica accessions from East Anatolia, Syria and Iraq were var. polyathera. Thirdly, the typica F1 hybrids between the Aegean and the Syrian typica accessions were backcrossed to the latter. Four of the seven BC1F1 plants obtained were var. typica, but the other three were var. polyathera. Based on these results, the following two conclusions were made. First, the awnless lateral spikelets characteristic of var. typica are due to two different genotypes: one is a dominant allele suppressing awn development on lateral spikelets and the other is a recessive allele(s) for awnless lateral spikelets with no dominant suppressor allele. Secondly, the former genotype occurs only in the western region of the distribution area of the species, while the latter occurs in the eastern region. The present results and the recent palaeopalynological evidence also suggested that var. polyathera, with more awns than var. typica, rapidly colonized Central Anatolia from the Levant or East Taurus/Zagros mountains arc after the last glacial period.
In this study, we determined the complete nucleotide sequence of the mitochondrial genome of the Japanese pond frog Rana nigromaculata. The length of the sequence of the frog was 17, 804 bp, though this was not absolute due to length variation caused by differing numbers of repetitive units in the control regions of individual frogs. The gene content, base composition, and codon usage of the Japanese pond frog conformed to those of typical vertebrate patterns. However, the comparison of gene organization between three amphibian species (Rana, Xenopus and caecilian) provided evidence that the gene arrangement of Rana differs by four tRNA gene positions from that of Xenopus or caecilian, a common gene arrangement in vertebrates. These gene rearrangements are presumed to have occurred by the tandem duplication of a gene region followed by multiple deletions of redundant genes. It is probable that the rearrangements start and end at tRNA genes involved in the initial production of a tandemly duplicated gene region. Putative secondary structures for the 22 tRNAs and the origin of the L-strand replication (OL) are described. Evolutionary relationships were estimated from the concatenated sequences of the 12 proteins encoded in the H-strand of mtDNA among 37 vertebrate species. A quartet-puzzling tree showed that three amphibian species form a monophyletic clade and that the caecilian is a sister group of the monophyletic Anura.
Using Escherichia coli strain VS101, whose hemH gene encoding the ferrochelatase is partially defective, we isolated and analyzed a clone (designated λ WH-1) from a λ phage library of soybean (Glycine max) cDNA, which exhibited weak complementation activity against the light sensitivity of VS101. In VS101 bacteria lysogenized with λWH-1, a significant decrease in accumulation of protoporphyrin IX (PROTO IX) was detected as compared with that in non-lysogenic bacteria. On the other hand, in the wild-type E. coli strains lysogenized with λ WH-1, significant accumulation of δ-aminolevulinic acid (ALA) was observed, although accumulation of other intermediates such as uroporphyrinogen III (UROGEN III) and coproporphyrinogen III (COPROGEN III), was not observed. The growth of the wild-type bacteria in which the insert cDNA from λWH-1 had been introduced via a plasmid vector was markedly inhibited. By constructing, testing and sequencing a series of deletion clones of the insert, it was found that the insert encodes two proteins, a trancated LepA and a hypothetical protein ORF296, and that only ORF296 possesses the ability to block the heme biosynthetic pathway. ORF296 showed about 30% identity with the E. coli hypothetical protein YicL. By cloning and examining the gene for YicL in E. coli, we found that YicL shows the same effect as that of the soybean cDNA. From these findings, we concluded that the clone from soybean and yicL from E. coli block a step in an early stage of the heme biosynthetic pathway (probably the step catalyzed by HemB). Consequently, we postulate that the VS101 bacteria harboring these genes became light resistant as a result of a decrease in accumulated PROTO IX, and that the growth of the bacteria harboring these genes was inhibited because of the inhibition of heme biosynthesis at the step catalyzed by HemB.
An uncharacterized gene, YNL078W, was isolated by the two-hybrid screening method using SHS1 (one of the septin genes) as bait and designated NIS1 (Neck protein Interacting with Septins). Nis1 interacts with all septins in the two-hybrid assay system. Physical interaction between Nis1 and Shs1 in vivo was confirmed by a co-immunoprecipitation experiment. Neither disruption nor overexpression of NIS1 caused a prominent phenotypic change. NAP1 was isolated by two-hybrid screening using NIS1 as bait. We detected physical interaction between Nis1 and Nap1 in vivo by a co-immunoprecipitation experiment. Nis1 was found to bind Gin4 and Kcc4 in the two-hybrid assay. Thus, a number of the proteins interacting with Nis1 are members of the mitotic signaling network. The stable maintenance of Nis1 was dependent on Nap1. Nis1 was phosphorylated throughout the cell cycle and was less abundant in G2/M phase. GFP-Nis1 is localized in the nucleus throughout the cell cycle and in the bud neck at G2/M phase in a septin-dependent manner. Altogether, the findings suggest that Nis1 may play a non-essential role in the mitotic signaling network.