The variation in CDKN2A mutations for the four features across continents is consistent with the lower melanoma incidence rates in Europe and higher rates of sporadic melanoma in Australia. The lack of a pancreatic cancer-CDKN2A mutation relationship in Australia probably reflects the divergent spectrum of mutations in families from Australia versus those from North America and Europe. GenoMEL is exploring candidate host, genetic and/or environmental risk factors to better understand the variation observed.

GenoMEL, comprising major familial melanoma research groups from North America, Europe, Asia, and Australia has created the largest familial melanoma sample yet available to characterize mutations in the high-risk melanoma susceptibility genes CDKN2A/alternate reading frames (ARF), which encodes p16 and p14ARF, and CDK4 and to evaluate their relationship with pancreatic cancer (PC), neural system tumors (NST), and uveal melanoma (UM). This study included 466 families (2,137 patients) with at least three melanoma patients from 17 GenoMEL centers. Overall, 41% (n = 190) of families had mutations; most involved p16 (n = 178). Mutations in CDK4 (n = 5) and ARF (n = 7) occurred at similar frequencies (2-3%). There were striking differences in mutations across geographic locales. The proportion of families with the most frequent founder mutation(s) of each locale differed significantly across the seven regions (P = 0.0009). Single founder CDKN2A mutations were predominant in Sweden (p.R112_L113insR, 92% of family's mutations) and the Netherlands (c.225_243del19, 90% of family's mutations). France, Spain, and Italy had the same most frequent mutation (p.G101W). Similarly, Australia and United Kingdom had the same most common mutations (p.M53I, c.IVS2-105A>G, p.R24P, and p.L32P). As reported previously, there was a strong association between PC and CDKN2A mutations (P < 0.0001). This relationship differed by mutation. In contrast, there was little evidence for an association between CDKN2A mutations and NST (P = 0.52) or UM (P = 0.25). There was a marginally significant association between NST and ARF (P = 0.05). However, this particular evaluation had low power and requires confirmation. This GenoMEL study provides the most extensive characterization of mutations in high-risk melanoma susceptibility genes in families with three or more melanoma patients yet available. (Cancer Res 2006; 66(20): 9818-28)

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Approximately 5% to 10% of melanoma may be hereditary in nature, and about 2% of melanoma can be specifically attributed to pathogenic germline mutations in cyclin-dependent kinase inhibitor 2A (CDKN2A). To appropriately identify the small proportion of patients who benefit most from referral to a genetics specialist for consideration of genetic testing for CDKN2A, we have reviewed available published studies of CDKN2A mutation analysis in cohorts with invasive, cutaneous melanoma and found variability in the rate of CDKN2A mutations based on geography, ethnicity, and the type of study and eligibility criteria used. Except in regions of high melanoma incidence, such as Australia, we found higher rates of CDKN2A positivity in individuals with 3 or more primary invasive melanomas and/or families with at least one invasive melanoma and two or more other diagnoses of invasive melanoma and/or pancreatic cancer among first- or second-degree relatives on the same side of the family. The work summarized in this review should help identify individuals who are appropriate candidates for referral for genetic consultation and possible testing.

The origin of plasmid DNA transfer, oriT, has been localized on RK2, a conjugative drug-resistance plasmid of the IncP group with a very broad host range in gram-negative bacteria. The transfer origin is contained in a 760-base-pair Hae II restriction fragment that maps in the same region as the singlestrand nick made by the RK2 relaxation complex. The functional oriT was subcloned as a 112-base-pair Hpa II fragment, and the DNA sequence of this region was determined. The dominant structural feature of the oriT sequence is a 19-base-pair inverted repeat, with 15 of the 19 bases able to form pairs in a hairpin structure. This inverted repeat may be the recognition site for the relaxation complex proteins, which nick the plasmid DNA molecule and initiate the transfer process.The transfer of plasmid DNA during bacterial conjugation begins at a specific site on the plasmid DNA molecule: the origin of transfer, oriT. The transfer origin was initially defined genetically as a site required in cis for plasmid transfer during mating (1, 2). Studies with the F plasmid of Escherichia coli have shown that a specific single strand of the DNA molecule is transferred with a leading 5' end (3, 4). DNA transfer is initiated by a strandand site-specific nick at oriT. The oriT of the F plasmid has been mapped near the start of the F transfer operon, within a 385-base-pair (bp) DNA fragment, and an oriT-specific nicking activity is encoded by the traY and traZ gene products (5, 6). The discovery of plasmid DNA-protein relaxation complexes has facilitated the study of the nicking process in other plasmids. Many bacterial plasmids can be isolated in the form of a superhelical DNA-protein relaxation complex, including R factors and colicinogenic plasmids (7-9). Upon treatment in vitro with detergents, proteases, or ethidium bromide, the supercoiled DNA is nicked in one strand and converted to the open circular form, a process called relaxation. In the case of the ColEl plasmid, the nick occurs at a specific site within a 320-bp region required in cis for transfer, the presumed oriT (10). A 60,000-dalton protein remains covalently attached to the 5' end of the nicked strand and probably facilitates transfer to the recipient cell (11-13). The ColEl plasmid is not self-transmissible and requires the presence of a conjugative plasmid in the same cell for mobilization during mating. However, study of the selftransmissible, broad host range IncP plasmid RK2 has revealed a similar property of the relaxation complex: the site-specific nicking event occurs within a 5-kilobase (kb) region of the plasmid that also contains oriT (14). In this paper, oriT of RK2 is localized to a 760-bp region that coincides with the relaxation nick. The functional oriT is contained within a 112-bp fragment, and the DNA sequence of this region has been determined. MATERIALS AND METHODSBacterial Strains and Plasmids. The E. coli strains used were HB101 (pro, leu, thi, lacY, strR, hsr-, hsm-, endoI-, recA), C600 Mu' (thr, leu, thi, SupE46, lacY, tonA), and...

BackgroundCopy number variation (CNV) has been recently identified in human and other mammalian genomes, and there is a growing awareness of CNV's potential as a major source for heritable variation in complex traits. Genomic selection is a newly developed tool based on the estimation of breeding values for quantitative traits through the use of genome-wide genotyping of SNPs. Over 30,000 Holstein bulls have been genotyped with the Illumina BovineSNP50 BeadChip, which includes 54,001 SNPs (~SNP/50,000 bp), some of which fall within CNV regions.ResultsWe used the BeadChip data obtained for 912 Israeli bulls to investigate the effects of CNV on SNP calls. For each of the SNPs, we estimated the frequencies of occurrence of loss of heterozygosity (LOH) and of gain, based either on deviation from the expected Hardy-Weinberg equilibrium (HWE) or on signal intensity (SI) using the PennCNV "detect" option. Correlations between LOH/CNV frequencies predicted by the two methods were low (up to r = 0.08). Nevertheless, 418 locations displayed significantly high frequencies by both methods. Efficiency of designating large genomic clusters of olfactory receptors as CNVs was 29%. Frequency values for copy loss were distinguishable in non-autosomal regions, indicating misplacement of a region in the current BTA7 map. Analysis of BTA18 placed major quantitative trait loci affecting net merit in the US Holstein population in regions rich in segmental duplications and CNVs. Enrichment of transporters in CNV loci suggested their potential effect on milk-production traits.ConclusionsExpansion of HWE and PennCNV analyses allowed estimating LOH/CNV frequencies, and combining the two methods yielded more sensitive detection of inherited CNVs and better estimation of their possible effects on cattle genetics. Although this approach was more effective than methodologies previously applied in cattle, it has severe limitations. Thus the number of CNVs reported here for the Holstein breed may represent as little as one-tenth of inherited common structural variation.

Two rRNA operons of Halobacterium marismortui were identified and cloned into plasmid pBR322 as 10-and 20-kilobase-pair (kbp) HindIII fragments, respectively. Restriction maps of the 10-kbp clone (pHH10) and an 8-kbp HindIII-ClaI subclone (pHC8) of the other operon were established. Southern hybridization of 16S, 23S, and 5S rRNA probes to the clones demonstrated that both operons code for the three rRNA species. By Sl nuclease analysis, the transcription initiation sites, some of the processing sites within the primary transcripts, and the boundaries of the mature 16S and 23S rRNA molecules were determined. Both operons are transcribed in vivo. Comparison of the two operons indicated that they are not identical. The most striking difference between the operons is the existence of three putative transcription initiation sites in one operon (HC8) and only one such site in the other operon (1H110). The regions surrounding these 5' transcript end sites share a high level of sequence similarity to each other and to the rRNA promoter regions of other halophilic archaebacteria. Analysis of the proximal 130 nucleotides of the two 16S rRNA genes indicated greater-than-expected sequence heterogeneity. There are a 2-base-pair insertion in the HC8 16S gene and 10 additional sites of nucleotide sequence heterogeneity.Ribosomes are an indispensable component of the protein synthesis apparatus. Because of its universality and structural conservation, the RNA component of the small ribosome subunit has proven to be an important and useful molecular chronometer for quantitating evolutionary relationships between organisms. Measurements based on these rRNA sequence comparisons have led Woese and Olsen to suggest that organisms can be arranged into three phylogenetic groups: eubacteria, archaebacteria, and eucaryotes.Each group represents a separate line of evolutionary descent from a common primordial ancestor (16,17).Within the archaebacteria, the genomic copy numbers of rRNA genes in different species range from one to four. The rRNA gene order is 5'-16S-23S-5S-3', except in sulfurdependent thermoacidophiles, in which the 5S gene is not linked to the 16S and 23S genes (2,7,8,10,11,14; for a review, see reference 4). In several halophilic and methanogenic organisms, an alanine tRNA is present in the 16S-23S intergenic space, and in the halophiles, a cysteine tRNA gene is present distal to the 5S gene (2,4,(7)(8)(9). When there is more than one operon, the tRNA genes are not always present in all copies.In Halobacterium cutirubrum, there is a single rRNA operon that contains the spacer alanine tRNA and the distal cysteine tRNA genes (7). Expression of this operon is driven by a series of five tandemly arranged promoters that operate with different efficiencies and exhibit growth rate-dependent regulation (3). A second species, H. marismortui, contains two separate rRNA operons. In this communication, we describe the cloning and initial characterization of these two operons. In many features, the two operons appear to differ both...

The transfer systems of broad host range IncP plasmids are increasingly used in the genetic analysis and manipulation of many gram-negative bacteria. We have examined the structural and functional relatedness of the transfer origins of ten different broad host range plasmids which belong to the IncP incompatibility group. The data reported here, together with our results on relatedness of the replication segments of these plasmids, demonstrate that the genomes of all IncP plasmids share extensive sequence homology in the regions specifying the transfer origin and replication functions. The homology results reveal the existence of two subclasses among IncP plasmids, designated here as IncP alpha and IncP beta. Furthermore, a functional analysis of the transfer origins of IncP plasmids suggests strongly that the DNA-nicking relaxation complex (Guiney and Helinski 1979) is required for plasmid transfer during conjugation.