Previously we analysed overlapping homozygous deletions in lung and breast tumours/tumour lines and de®ned a small region of 120 kb (part of LCTSGR1) at 3p21.3 that contained putative lung and breast cancer tumour suppressor gene(s) (TSG). Eight genes including RASSF1 were isolated from the minimal region. However, extensive mutation analysis in lung tumours and tumour lines revealed only rare inactivating mutations. Recently, de novo methylation at a CpG island associated with isoform A of RASSF1 (RASS-F1A) was reported in lung tumours and tumour lines. To investigate RASSF1A as a candidate TSG for various cancers, we investigated: (a) RASSF1A methylation status in a large series of primary tumour and tumour lines; (b) chromosome 3p allele loss in lung tumours and (c) RASSF1 mutation analysis in breast tumours. RASSF1A promoter region CpG island methylation was detected in 72% of SCLC, 34% of NSCLC, 9% of breast, 10% of ovarian and 0% of primary cervical tumours and in 72% SCLC, 36% NSCLC, 80% of breast and 40% of ovarian tumour lines. In view of the lower frequency of RASSF1 methylation in primary breast cancers we proceeded to RASSF1 mutation analysis in 40 breast cancers. No mutations were detected, but six single nucleotide polymorphisms were identi®ed. Twenty of 26 SCLC tumours with 3p21.3 allelic loss had RASSF1A methylation, while only six out of 22 NSCLC with 3p21.3 allele loss had RASSF1A methylation (P=0.0012), one out of ®ve ovarian and none out of six cervical tumours with 3p21.3 loss had RASSF1A methylation. These results suggest that (a) RASSF1A inactivation by two hits (methylation and loss) is a critical step in SCLC tumourigenesis and (b) RASSF1A inactivation is of lesser importance in NSCLC, breast, ovarian and cervical cancers in which other genes within LCTSGR1 are likely to be implicated. Oncogene (2001) 20, 1509 ± 1518.

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The sector of the genome of broad-host-range lncP plasmid RK2 from kb coordinate 5 4 0 to 600 confers an active partitioning phenotype, increasing the segregational stability of low-copy-number unstable plasmids. This Par region encodes the central control operon (korA, incC, korB, korF and korG) and the associated genes kfrA, upf54.8 and upf54.4. Each ORF in this region was knocked out in turn and it was shown that only incC and korB are needed for the stability phenotype. incC encodes two polypeptides from alternative translational starts. A deletion of the start of the operon showed that only IncC2, the shorter product, is essential for partitioning. Directed mutation or deletion was used to inactivate in turn each of the three KorB-binding sites (0,s) which were candidate cis-acting sequences needed for stability. Only inactivation of OB3, which lies between upf54.4 and upf54.8, resulted in an increased rate of segregational loss. However, the rate of loss was significantly higher than the rate of loss of the test plasmid carrying none of this RK2 Par region. Either inactivation of korB or deletion of OBI from this OB3 mutant resulted in restoration of the loss rate to that expected for the unstable test plasmid alone. Thus KorB can act on OBI to create a complex that either inhibits replication or reduces the effective plasmid copy number, perhaps by promoting pairing between plasmid molecules. This implies that RK2 goes through a cycle of pairing and separation, akin to the mitotic cycle of eukaryotic chromosomes.

No abstract

SummaryKorB protein (358 amino acids) binds to 12 highly conserved sequences on the RK2 genome and co-ordinates the expression of at least five operons encoding genes for stable inheritance and plasmid transfer. KorB represses the trfA, korA and klaA promoters where it binds 4 bp upstream of the ¹35 region (class I KorB operators, O B ). We show here that KorB on its own can also repress the trbA, trbB, kfrA and kleA promoters where O B is between 80 and 189 bp away from the transcription start point (class II operator). A C-terminal deletion of 17 amino acids resulted in the loss of KorB's ability to repress through class II operator but not through class I operator. This deletion reduced multimerization of His 6 -tailed KorB protein in vitro and greatly reduced binding specificity for fragments containing O B sequences. At the trbBp region, where O B 9 lies 189 bp upstream of the transcription start point, mutagenesis of a proposed secondary binding site overlapping the trbBp ¹35 region had no effect on the ability of KorB to repress trbBp. Nevertheless, gel retardation analysis showed that KorB binding is promoted by sequences upstream and downstream of O B 9 and that KorB can form higher order complexes on DNA. However, DNase I footprinting suggested that RNA polymerase may interact directly with KorB bound at O B 9 and implied that contacts between these proteins could be responsible for the action of KorB at a distance.