Construction of consecutive deletions of the Escherichia coli chromosome

The minimal set of genetic information necessary and sufficient to sustain a functioning cell contains not only trans-acting genes, but also cis-acting chromosomal regions that cannot be complemented by plasmids carrying these regions. In Escherichia coli (E. coli), only one chromosomal region, the origin of replication has been identified to be cis-acting. We constructed a series of mutants with long-range deletions, and the chromosomal regions containing trans-acting essential genes were deleted in the presence of plasmids complementing the deleted genes. The deleted regions cover all regions of the chromosome except for the origin and terminus of replication. The terminus affects cell growth, but is not essential. Our results indicate that the origin of DNA replication is the only vital, unique cis-acting DNA sequence in the E. coli chromosome necessary for survival

加温後のtsAF8細胞の細胞周期

Thermotolerance in tsAF8 cells develops during incubation at 34℃ after heating at 45℃, while it is suppressed by the following incubation at a non-permissive temperature of 39.7℃ after the same heating. The incubation temperature after heating may affect the cell cycle and consequently thermotolerance. In the present study, a relationship between the thermotolerance and the cell cycle of tsAF8 was investigated. The cell cycle fractions and DNA synthesis were measured by flow cytometry using double staining with propidium iodide and bromodeoxyuridine. When the tsAF8 cells were heated at 45℃ for 20 min, and thereafter incubated at 34℃, bromodeoxyuridine uptake in the S phase cells (DNA synthesis) was recovered to 65.1% 6 h after the heating, and the cells showed gradual accumulation in the G(2)/M phase. When the cells were incubated at 39.7℃ after heating at 45℃ for 20 min, then showed inhibition of thermotolerance development, the DNA synthesis was recovered to 15.1% temporarily 6 h after the heating, but it became 0% after 12 h, and the cells did not remarkably accumulate in any phases of the cell cycle. This inhibition of DNA synthesis at 39.7℃ was considered to be the result of cell survival decreasing by a step-down heating. However, the relationship between the thermotolerance and the cell cycle was not found out in tsAF8 cells, because the cells did not accumulate in any phases of the cell cycle under the inhibitory condition of thermotolerance.tsAF8細胞は45℃の加温後34℃で培養すると温熱耐性が速やかに発現するが,加温後,制限温度である39.7℃で培養すると温熱耐性の発現が抑制される｡加温後の培養温度が細胞周期に影響し,その結果として温熱耐性発現に影響を与えている可能性があることから,今回,Propidium Iodide(PI)とbromodeoxyuridine(BrdU)でtsAF8細胞を二重染色し,フローサイトメトリーによって温熱耐性と細胞周期の関係の有無について調べた｡tsAF8細胞を45℃20分の加温後34℃で培養すると,6時間後にはG(1)期の細胞が減少し,12時間後にはG(2)/M期への蓄積が見られた｡しかし,加温後39.7℃で培養した場合には細胞周期の進行がほとんど見られなかった｡BrdU の取込みは,加温せずに39.7℃で培養した場合には活発に行われ,また,45℃20分加温後34℃で培養した場合には,6時間後にはBrdUの取り込みは65.1%まで回復した｡しかし,温熱耐性発現の抑制が観察される45℃20分加温後39.7℃で培養した場合には,BrdUの取込み量は6時間後に一時的に15.1%に回復するが,12時間後には取込み量はゼロとなった｡BrdUの取り込みが阻害されたのはstep-down heatingの現象による細胞生存率の減少が原因だと考えられたが,温熱耐性発現の抑制が観察される条件下では細胞周期の特定の時期への集積がなかったことから,温熱耐性と細胞周期との関係はtsAF8細胞においては見い出されなかった

A point mutation found in the WT1 gene in a sporadic Wilms' tumor without genitourinary abnormalities is identical with the most frequent point mutation in Denys-Drash syndrome

AbstractWe have analyzed exon 9 of the WT1 gene of 18 non-familial/sporadic unilateral Wilms' tumors (WTs) from Japanese patients, by the polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) method. After screening these WTs, a nucleotide alternation, which was present on both alleles, was found in only one case. Furthermore, PCR-SSCP analysis of the constitutional DNA revealed that this patient carried the mutation on only one allele in the germline. Sequence analysis showed that the tumor carried a point mutation (C-1180 to T-1180) in WT1 exon 9 of both alleles, resulting in an Arg-394 to Trp-394 amino acid substitution within the third zinc finger domain of the WT1 product. Interestingly, this mutation is identical with the most frequent point mutation associated with the Denys-Drash syndrome. However, the classical triad of Denys-Drash syndrome does not apply to this patient. This is in the first report of the point mutation in the zinc finger domain of both WT1 alleles in a sporadic unilateral WT without genitourinary abnormalities, and the mutation suggests that some sporadic WTs carry the Denys-Drash WT1 mutations

Mutations identified in engineered Escherichia coli with a reduced genome

Characterizing genes that regulate cell growth and survival in model organisms is important for understanding higher organisms. Construction of strains harboring large deletions in the genome can provide insights into the genetic basis of cell growth compared with only studying wild-type strains. We have constructed a series of genome-reduced strains with deletions spanning approximately 38.9% of the E. coli chromosome. Strains were constructed by combining large deletions in chromosomal regions encoding nonessential gene groups. We also isolated strains Δ33b and Δ37c, whose growth was partially restored by adaptive laboratory evolution (ALE). Genome sequencing of nine strains, including those selected following ALE, identified the presence of several Single Nucleotide Variants (SNVs), insertions, deletions, and inversions. In addition to multiple SNVs, two insertions were identified in ALE strain Δ33b. The first was an insertion at the promoter region of pntA, which increased cognate gene expression. The second was an insertion sequence (IS) present in sibE, encoding the antitoxin in a toxin-antitoxin system, which decreased expression of sibE. 5 strains of Δ37c independently isolated following ALE harboring multiple SNVs and genetic rearrangements. Interestingly, a SNV was identified in the promoter region of hcaT in all five strains, which increased hcaT expression and, we predict, rescued the attenuated Δ37b growth. Experiments using defined deletion mutants suggested that hcaT encodes a 3-phenylpropionate transporter protein and is involved in survival during stationary phase under oxidative stress. This study is the first to document accumulation of mutations during construction of genome-reduced strains. Furthermore, isolation and analysis of strains derived from ALE in which the growth defect mediated by large chromosomal deletions was rescued identified novel genes involved in cell survival