Single nucleotide polymorphism in prohibitin 3' untranslated region and breast-cancer susceptibility.

The RNA encoded by the 3' untranslated region of the prohibitin gene arrests cell proliferation by blocking the transition between the G1 and S phases of the cell cycle. The product of a variant allele (T allele) is inactive. We did a case-control study of prohibitin genotype in 205 women with breast cancer and 1046 healthy controls. The results showed an association between the T allele and breast cancer in women who reported a first-degree relative with the disease (odds ratio 2.5, p=0.005). An even stronger association was found in a subset of women diagnosed at or before age 50 years (4.8, p=0.003). These data suggest that prohibitin genotyping has value in assessing risk of breast cancer in women aged 50 years or younger with at least one first-degree relative with the disease.

Prohibitin in breast cancer cell lines: loss of antiproliferative activity is linked to 3' untranslated region mutations.

The evolutionarily conserved prohibitin gene is located on human chromosome 17q21, and two alleles have been identified. Our previous studies characterizing prohibitin in immortalized cells, classified into four complementation groups (A-D) based on the ability of whole-cell hybrids to become senescent, have suggested that it has tumor suppressor activity in group B cells. Only the cell lines assigned to group B are sensitive to the antiproliferative activity of prohibitin, and all are homozygous for an allele designated B because of its exclusive association with this group. Prohibitin genotyping of 22 breast cancer cell lines identified 17 homozygous for the B allele, 5 homozygous for the non-B allele, and no heterozygotes. Four of these cell lines were chosen for further characterization of prohibitin. In cell proliferation assays, the homozygous B breast cancer cell lines (BT-20, SK-BR-3, and MCF7) are all inhibited from traversing the cell cycle following the introduction of wild-type prohibitin transcripts. The cell line homozygous for the alternative non-B allele (BT-549) is not inhibited by transcripts. All of the breast cancer cell lines overexpress the longer form of the prohibitin mRNA (1.9 kb) and the protein. Mutational analysis of the protein-coding region detected no mutations in any of the lines. However, BT-20, SK-BR-3, and MCF7 cells are all mutated in the final 200 bases of the 3' untranslated region (3'UTR) exclusive to the 1.9-kb transcript, but BT-549 cells had no alterations in this region of the 3'UTR. Functional mapping experiments performed in the mutated SK-BR-3 line showed that the wild-type 3'UTR alone is sufficient to inhibit cell cycle progression, indicating that the antiproliferative activity of the prohibitin transcript is localized to this region. Overall, our results show that most (80%) of the cell lines derived from breast tumors have a common prohibitin genotype, suggesting that they belong to the same group of immortalized cells, group B. The results also show that the prohibitin 3'UTR exhibits the characteristics of a trans-acting regulatory RNA (riboregulator), the tumor suppressor activity of which is inactivated by mutation in group B immortalized cells.

The 3' untranslated region of prohibitin and cellular immortalization.

We have been studying the role of the evolutionarily conserved prohibitin gene in cellular immortalization and tumor suppression. Immortalized human cells are classified into four complementation groups (A, B, C, and D) based on the ability of fusion hybrids to become senescent. The present study expands our preliminary evidence showing that the antiproliferative activity of prohibitin is only effective in immortalized Group B cells and normal cells. Data presented here show that the expression of a prohibitin mRNA with a long 3' untranslated region (3'UTR) and prohibitin protein is elevated in immortalized cells from all complementation groups. However, all immortalized cells classified in complementation Group B, and no cell lines in any of the other groups, are sensitive to the antiproliferative activity of wild-type prohibitin transcripts. All Group B cells are also homozygous for one of two human prohibitin alleles that are distinguishable by two distinct intron polymorphism restriction sites. Interestingly, sequence analysis of the prohibitin gene from representatives of each of the complementation groups showed that the 3'UTR from Groups A, C, and D matched wild type; however, the sequence from all four Group B cell lines differed from wild type. Functional inhibition assays on truncated wild-type mRNA transcripts as well as 3'UTR specific wild-type and mutated transcripts show that the antiproliferative activity of prohibitin resides, at least in part, in the 3'UTR. These data suggest that the prohibitin 3'UTR may function as a trans-acting regulatory RNA (riboregulator) whose tumor suppressor activity has been inactivated by mutation in Group B cells.

Prohibitin and the senescent phenotype.

Prohibitin is an evolutionarily conserved gene that has antiproliferative activity, is ubiquitously expressed, and appears to be essential for cell survival. The gene codes for a 30 kD, post-synthetically modified protein located primarily in the mitochondria. It functionally inhibits cell cycle traverse and DNA synthesis, but its mechanism of action is presently unknown. Prohibitin is proposed to be a member of a new class of tumor suppressor genes whose inhibitory activity plays a role in the dominant senescent phenotype. Its involvement in senescence has been postulated from results obtained from such diverse systems as yeast and human diploid fibroblasts. Additional data show that prohibitin is involved in one of the limited number of pathways that results in the loss of the senescent phenotype and leads to cellular immortalization. Its involvement, however, occurs downstream in the pathway and is postulated to be part of the lost tumor suppressor activities associated with tumorigenicity.

Prohibitin antiproliferative activity and lack of heterozygosity in immortalized cell lines.

Experiments were performed to determine whether prohibitin, an evolutionarily conserved gene with antiproliferative activity, has a role in cellular immortalization. A cell proliferation assay was used to examine one human cell line from each of four established immortal complementation groups, termed A, B, C, and D, and a normal human diploid fibroblast line. Only normal and Group B cells were inhibited from traversing the cell cycle after introduction of wild-type prohibitin transcript. All of the immortalized cells expressed elevated levels of prohibitin mRNA and protein. Prohibitin gene structural characterization using Southern and single-strand conformation polymorphism (SSCP) analyses distinguished two alleles. One is cleaved at a polymorphic intronic EcoRI site, exhibits an exon 6-associated SSCP, and is homozygous only in Group B cells. The other is not cleaved at the EcoRI site, has a different exon 6 SSCP pattern, and is homozygous in Groups A, C, and D. In contrast, normal cells are heterozygous for the alleles. These results suggest that prohibitin may play a role as a tumor suppressor in the immortalization of Group B cells.

Regions of evolutionary conservation between the rat and human prohibitin-encoding genes.

We have analyzed and compared the 5' promoter region, the intron structure and the exon-intron flanking sequences in the rat and human prohibitin-encoding genes (PHB). Comparative analysis of a 350-nt region immediately 5' to and including the first exon identifies eight highly conserved regions, four of which correspond to binding sites for known transcriptional control proteins (CCAAT box, 'SV40' site and two Sp1 sites). The promoter lacks a TATA box. Four transcription start points (tsp) clustered within a 35-bp region were identified by rapid amplification of cDNA ends (RACE). The exon-intron boundaries in rat and human are highly conserved, with identical positioning of splice junctions. PCR analysis with conserved exon primers was used to detect length variation between rat and human PHB, and length differences were observed in all of the introns.

Prohibitin: potential role in senescence, development, and tumor suppression.

Prohibitin is an evolutionarily conserved gene with homologues found in organisms ranging from yeast to man. In man the gene is located on chromosome 17 at q21. The deduced amino acid sequences of the protein products from mouse and rat are identical; and these differ from the human protein sequence by a single conserved amino acid. Prohibitin has antiproliferative activity and available data suggest a role in such diverse processes as normal cell cycle regulation, replicative senescence, cellular immortalization, and the development of sporadic breast tumors. Although its functional activity is presently unknown, the 30,000-Da protein has been located in the inner membrane of mitochondria, where it is postsynthetically modified, as well as on the plasma membrane of B cells, where it is associated with the IgM receptor. Prohibitin's evolutionary conservation and ubiquitous expression indicate that it is a fundamentally important gene; and current data suggest a functional role in such dissimilar processes as development, senescence, and tumor suppression.

Specialized chromatin structure domain boundary elements flanking a Drosophila heat shock gene locus are under torsional strain in vivo.

An in vivo assay employing psoralen cross-linking was used to investigate the presence of unrestrained supercoiling in DNA sequences located in nontranscribed regions flanking the 3' ends of the pair of divergent heat shock protein 70 (hsp70) genes at locus 87A7 of Drosophila. Two of the regions examined contain sequences comprising the previously defined specialized chromatin structure elements (scs and scs'). Both of these putative chromosomal domain boundaries exhibited very similar levels of unrestrained negative supercoiling that remained high regardless of the transcriptional status of the hsp70 genes. The steric accessibility of the scs region before heat shock was 3-fold higher than either flanking region (consistent with its previously documented DNase I hypersensitivity); this increased an additional 2-fold following hsp70 gene activation without a concomitant rise in the accessibility of flanking regions. Most notably, a sequence which lies outside the presumed 87A7 domain, as defined by the centromere-proximal scs element, exhibited no detectable torsional tension regardless of gene activity in the domain. A sequence located just inside the scs region displayed a low level of tension that was also essentially unaffected by transcription, consistent with data obtained previously for a similarly situated fragment at the centromere-distal scs' location. The existence of a highly localized region of supercoiling within the scs and scs' sequences might be related to their activity in vivo as insulators of chromosomal position effects in Drosophila.

Stably maintained microdomain of localized unrestrained supercoiling at a Drosophila heat shock gene locus.

A psoralen crosslinking assay was utilized to detect localized, unrestrained DNA supercoiling (torsional tension) in vivo in Drosophila chromosomal regions subject to differential transcriptional activity. By comparing rates of crosslinking in intact cells with those in cells where potential tension in chromosomal domains was relaxed by DNA strand nicking, the contribution to psoralen accessibility caused by altered DNA-protein interactions (e.g. nucleosomal perturbations) was distinguished from that due to the presence of unrestrained supercoiling in a region of interest. The heat shock protein 70 (hsp70) genes were wound with a significant level of superhelical tension that remained virtually unaltered whether or not the genes were transcriptionally activated by thermal elevation. Constitutively expressed 18S ribosomal RNA genes also exhibited unrestrained superhelical tension at a level comparable with that across hsp70. In contrast, flanking regions downstream of each of the divergent hsp70 genes at locus 87A7 exhibited substantially less tension. Thus the results point to the existence of stable, torsionally stressed topological domains within eukaryotic chromosomal DNA, suggesting that the relaxing action of topoisomerases is not ubiquitous throughout the nucleus but, in fact, is likely to be tightly regulated.

DNaseI-sensitive and undermethylated rDNA is preferentially expressed in a maize hybrid.

An Eco RI polymorphism, present in the 26S ribosomal RNA gene (rDNA) of the maize hybrid Sx19 (B73 x Mo17), was utilized to correlate DNaseI sensitivity, undermethylation and expression in rDNA. We have previously shown that in double digest experiments with methylation-sensitive restriction enzymes and Eco RI, Sx19 rDNA fragments originating from repeat units with two Eco RI sites (8.0 kb) are undermethylated, whereas the fragments originating from repeat units with a single Eco RI site (9.1 kb) are completely methylated. In the present study, Sx19 rDNA chromatin structure was examined by purifying intact nuclei and digesting them briefly with increasing amounts of DNaseI. Analysis of this DNA with Eco RI showed that the 8.0 kb rDNA fragments are extremely sensitive to DNaseI digestion, while the 9.1 kb rDNA fragments are relatively resistant to digestion even at high levels of DNaseI. Specific sites hypersensitive to DNaseI cleavage were mapped to a region in the intergenic spacer (IGS) near the major undermethylated site. Analysis of polymerase chain reaction (PCR) products synthesized using Sx19, B73, and Mo17 DNAs as templates indicated that the Eco RI polymorphism is due to a base change in the recognition site. Direct rRNA sequencing identified a single-base change in Mo17 rRNA relative to B73 rRNA. Allele-specific oligonucleotide probes containing the region surrounding and including the Eco RI polymorphic site were utilized to detect a nucleolar dominance effect by quantitating levels of rRNA transcripts in Sx19 and the reciprocal cross. Results from these single-base-pair mismatch hybridization experiments indicate that the majority of the rRNA transcripts in Sx19 originate from the DNaseI-sensitive, undermethylated, Eco RI-polymorphic rDNA repeat units.

Unmethylated regions in the intergenic spacer of maize and teosinte ribosomal RNA genes.

The restriction endonucleases Hpa II and Msp I were used to examine cytosine methylation in the ribosomal RNA genes (rDNA) of inbred lines of maize and species of teosinte. In all of the rDNAs examined, Msp I (not sensitive to mCpG) digestion yielded a distribution of lower molecular weight fragments indicative of multiple recognition sites. The majority of the rDNA arrays in an individual were inaccessible to Hpa II (sensitive to mCpG) cleavage, but a significant fraction (10-25%) was cleaved at least once by Hpa II into repeat unit length fragments (9.1 kbp). In some maize inbred lines, one or two additional fragment populations (less than 9.1 kbp in length) were also produced by Hpa II digestion. All of the unmethylated Hpa II sites mapped to the intergenic spacer (IGS), and the major unmethylated site was located approximately 800 bp 5' to the start of the 18S RNA coding sequence. An Eco RI polymorphism, present in the 26S gene of certain inbred lines and hybrids, was utilized to investigate the organization of unmethylated repeat units in the rDNA array. In double digest experiments with Hpa II/Eco RI, the fragments from repeat units with two Eco RI sites were sensitive to Hpa II digestion, whereas, the fragments from repeat units with a single Eco RI site were almost completely resistant to Hpa II digestion. Similar digestion patterns were also observed in Eco RII (sensitive to mCNG)/Eco RI digests. These results suggest that unmethylated and Eco RI polymorphic sites occur in the same repeat units.

Ribosomal gene structure, variation and inheritance in maize and its ancestors.

We have examined the structure of nuclear genes coding for ribosomal RNAs in maize and its wild relatives, the teosintes and Tripsacum. Digestion of the rDNA (genes coding for 18S, 5.8S and 26S RNAs) with 15 restriction endonucleases (with six base pair recognition sites) yields essentially a single map for the approximately 10,000 repeat units within an individual plant or species. Both length and site variation were detected among species and were concentrated in the intergenic spacer region of the rDNA repeat unit. This result is in agreement with patterns of rDNA change observed among wheat and its relatives (Triticeae), and among vertebrate species. Digestion of these nuclear DNAs with BamHI and subsequent hybridization with a 5S RNA gene-specific probe allowed determination of the size of the 5S gene repeat unit in maize, teosintes, and Tripsacum. Groupings in the genus Zea were characterized by distinct repeat unit types five Tripsacum species examined shared a 260 base pair major repeat unit type. Additionally, several other restriction endonuclease cleavage patterns differentiated among the 5S DNAs within the genus Zea. The rDNA and 5S DNA restriction site variation among the species can be interpreted phylogenetically and agrees with biochemical, karyotypic, and morphological evidence that places maize closest to the Mexican teosintes. For both gene arrays, contributions from each parental genome can be detected by restriction enzyme analysis of progeny from crosses between maize and two distantly related teosintes, Zea luxurians or Zea diploperennis, but certain teosinte arrays were underrepresented in some of the hybrids.

Nuclear ribosomal RNA genes and algal phylogeny--the Chlamydomonas example.

The nuclear ribosomal RNA genes (rDNA) of Chlamydomonas reinhardtii, C. moewusii and C. eugametos were examined with restriction endonuclease fragment and direct rRNA sequencing analyses. These comparative molecular data confirm similarity between C. moewusii and C. eugametos, and dissimilarity between the strains and C. reinhardtii. For C. moewusii and C. eugametos, the fragment analysis of digests with 16 (six base pair recognition site) restriction endonucleases revealed either no or minor differences. These minor differences appear to be confined to length and site variation in the rapidly evolving intergenic spacer region of the algal rDNA repeat unit. In contrast, patterns of digests for C. reinhardtii were completely different from those of C. moewusii and C. eugametos for all enzymes tested. Over two regions of the 18S ribosomal RNA (spanning approx. 300 bases) in C. moewusii and C. eugametos, we observed three possible base substitutions and no insertion/deletion events. The same comparison between C. reinhardtii and C. moewusii (or C. eugametos) revealed 31 base substitutions and eight insertion/deletion events. Overall, the rDNA comparisons support the proposed conspecificity of C. moewusii and C. eugametos, as well as the hypothesis that intraspecific variation in the algal ribosomal RNA coding region is minimal and that comparisons of rDNA sequences at higher taxonomic levels can be useful indicators of algal phylogeny. The degree of difference in the sequences of the 18S coding region between C. reinhardtii and C. moewusii or C. eugametos is comparable to that between an angiosperm and Equisetum and may reflect an ancient divergence between two species in one algal genus.(ABSTRACT TRUNCATED AT 250 WORDS)

Stage-specific DNA methylation in a fungal plant pathogen.

Significantly more 5-methylcytosine residues were found in the DNA from the dormant sclerotia of Phymatotrichum omnivorum than in the DNA from the metabolically active mycelia of the fungus, as shown by high-pressure liquid chromatography of acid-hydrolyzed DNA digests and by restriction of the DNA with the isoschizomers MspI and HpaII. N6-Methyladenine was not detected in GATC sequences in the DNA isolated from either stage.