Variable MLH1 promoter methylation patterns in endometrial carcinomas of endometrioid subtype lacking DNA mismatch repair.

A lack of DNA mismatch repair (MMR) is observed in approximately 20% of sporadic endometrial tumors, but few of these tumors have mutations in MSH2 or MLH1, the two major MMR genes. Promoter methylation is an important means of silencing transcription, and methylation of the MLH1 promoter has been reported as an important cause of MLH1 inactivation in endometrial cancers. Studies have shown that specific CpG sites within the MLH1 gene promoter are critical for gene expression, but other studies have shown that methylation of both more proximal and more distal sequences are important for MLH1 gene regulation. Here, we used a microsatellite instability assay and MLH1 immunohistochemistry to identify a subset of endometrial carcinomas of the endometrioid subtype lacking MMR. Sequencing of bisulphite-treated DNA from these tumors determined the methylation status of 42 CpG sites across the MLH1 promoter (spanning -204 to -702 bp upstream of the transcriptional start). Unlike the 4 normal endometrial samples that were unmethylated, 17 of 21 MMR-deficient samples showed complete or near-complete methylation and the remaining 4 MMR-deficient samples had a considerable degree of methylation (approximately 50% or greater). Five tumors demonstrated isolated unmethylated CpG sites, despite methylation throughout the rest of the promoter. This underscores the importance of examining the methylation status of at least several CpG sites within the promoter as methylation is not always consistent across DNA. Overall, our findings support the model that density of methylation of CpG sites across the MLH1 promoter is important in determining gene expression.

The "flap" endonuclease gene FEN1 is excluded as a candidate gene implicated in the CAG repeat expansion underlying Huntington disease.

At least 12 disorders including Huntington disease (HD) are associated with expansion of a trinucleotide repeat (TNR). Factors contributing to the risk of expansion of TNRs and the mechanism of expansion have not been elucidated. Data from Saccharomyces cerevisiae suggest that the flap endonuclease FEN1 plays a role in expansion of repetitive DNA tracts. It has been hypothesized that insufficiency of FEN1 or a mutant FEN1 might contribute to the occurrence of expansion events of long repetitive DNA tracts after polymerase slippage events during lagging strand synthesis. The expression pattern of FEN1 was determined, and ubiquitous tissue expression, including germ cells, suggested that FEN1 has the potential to be involved in HD. Fifteen HD parent/child pairs that demonstrated intergenerational increases in CAG length of greater than 10 repeats were examined for possible mutations or polymorphisms within the FEN1 gene that could underlie the saltatory repeat expansions seen in these individuals. No alterations were observed compared to 50 controls, excluding FEN1 as a trans-acting factor underlying TNR expansion. The identification of a candidate gene(s) in HD or other CAG-expansion disorders implicated in TNR instability will elucidate the mechanism of expansion for this growing family of neurological disorders.

Somatostatin family of peptides and its receptors in fish.

Somatostatin (SRIF or SS) is a phylogenetically ancient, multigene family of peptides. SRIF-14 is conserved with identical primary structure in species of all classes of vertebrates. The presence of multiple SRIF genes has been demonstrated in a number of fish species and could extend to tetrapods. Three distinct SRIF genes have been identified in goldfish. One of these genes, which encodes [Pro2]SRIF-14, is also present in sturgeon and African lungfish, and is closely associated with amphibian [Pro2,Met13]SRIF-14 gene and mammalian cortistatin gene. The post-translational processing of SRIF precursors could result in multiple forms of mature SRIF peptides, with differential abundance and tissue- or cell type-specific patterns. The main neuroendocrine role of SRIF-14 peptide that has been determined in fish is the inhibition of pituitary growth hormone secretion. The functions of SRIF-14 variant or larger forms of SRIF peptide and the regulation of SRIF gene expression remain to be explored. Type 1 and type 2 SRIF receptors have been identified from goldfish and a type 3 SRIF receptor has been identified from an electric fish. Fish SRIF receptors display considerable homology with mammalian counterparts in terms of primary structure and negative coupling to adenylate cyclase. Although additional types of receptors remain to be determined, identification of the multiple gene family of SRIF peptides and multiple types of SRIF receptors opens a new avenue for the study of physiological roles of SRIF, and the molecular and cellular mechanisms of SRIF action in fish.

Dopaminergic regulation of three somatostatin mRNAs in goldfish brain.

Three distinct somatostatin cDNAs characterized previously from goldfish brain encode three preprosomatostatins (PSS), designated as PSS-I, PSS-II and PSS-III. In this study, dopaminergic regulation of PSS gene expression was examined by Northern blot analysis in the forebrain of goldfish. Intraperitoneal injection of the non-selective dopamine (DA) agonist, apomorphine, significantly decreased the levels of all three PSS mRNAs, indicating an inhibitory regulation of PSS gene expression by DA. The involvement of DA receptor subtypes in the regulation of PSS gene expression was examined using the D1 receptor agonist and antagonist drugs SKF 38393 and SCH 23390, and the D2 agonist and antagonist drugs LY 171555 and pimozide, respectively. The results provide evidence for inhibitory and/or stimulatory regulation of PSS gene expression by DA through both D1 and D2 receptors, which are dependent on the temporal pattern of dopamine input and reproductive stage of the fish. Demonstration of involvement of both DA D1 and D2 receptors in the dopaminergic regulation of goldfish brain PSS gene expression is a novel finding, distinct from the observations in mammalian models.

Expression of three distinct somatostatin messenger ribonucleic acids (mRNAs) in goldfish brain: characterization of the complementary deoxyribonucleic acids, distribution and seasonal variation of the mRNAs, and action of a somatostatin-14 variant.

In this study, three somatostatin (SRIF) complementary DNAs (cDNAs) were characterized from goldfish brain. The cDNAs encode three distinct preprosomatostatins (PSS), designated as PSS-I, PSS-II, and PSS-III. The goldfish PSS-I, PSS-II, and PSS-III contain enzymatic cleavage recognition sites, potentially yielding SRIF-14 with sequence identical to mammalian SRIF-14, SRIF-28 with [Glu1, Tyr7, Gly10]SRIF-14 at its C-terminus, and [Pro2]SRIF-14, respectively. The brain distribution of the three SRIF messenger RNAs (mRNAs) were differential but overlapping in the telencephalon, hypothalamus and optic tectum-thalamus regions. Seasonal variations in the levels of the three mRNAs were observed, with differential patterns between the three mRNAs and differences between the sexes. However, only the seasonal alteration in the levels of the mRNA encoding PSS-I showed close association with the seasonal variation in brain contents of immunoreactive SRIF-14 and inversely correlated with the seasonal variation in serum GH levels described in the previous studies, suggesting that SRIF-14 is involved in the control of the seasonal variation in serum GH levels. The putative SRIF-14 variant, [Pro2]SRIF-14, inhibited basal GH secretion from in vitro perifused goldfish pituitary fragments, with similar potency to SRIF-14; [Pro2]SRIF-14 also inhibited stimulated GH release from the pituitary fragments, supporting that [Pro2] SRIF-14 is a biologically active form of SRIF in goldfish.

Evolution of neuroendocrine peptide systems: gonadotropin-releasing hormone and somatostatin.

Nine vertebrate and two protochordate gonadotropin-releasing hormone (GnRH) decapeptides have been identified and sequenced. Multiple molecular forms of GnRH peptide were present in the brain of most species examined, and cGnRH-II generally coexists with one or more GnRH forms in all the major vertebrate groups. The presence of multiple GnRH forms has been further confirmed by the deduced GnRH peptide structure from cDNA and/or gene sequences in several teleost species and tree shrew. High conservation of the primary structure of GnRH decapeptides and the overall structure of GnRH genes and precursors suggests that they are derived from a common ancestor. Somatostatin (SRIF) is a phylogenetically ancient, multigene family of peptides. A tetradecapeptide, SRIF (SRIF14) has been conserved, with the same amino acid sequence, in representative species of all classes of vertebrate. Four molecular variants of SRIF14 have been identified. SRIF14 is processed from preprosomatostatin-I, which contains SRIF14 at its C-terminus; preprosomatostatin-I is also processed to SRIF28 in mammals and SRIF26 in bowfin. Teleost fish possess a second somatostatin precursor, preprosomatostatin-II, containing [Tyr7, Gly10]-SRIF14 at the C-terminus, that is mainly processed into large forms of SRIF.

Transferable Streptomyces DNA amplification and coamplification of foreign DNA sequences.

The 8.8-kb amplifiable unit of DNA of Streptomyces achromogenes subsp. rubradiris, AUD-Sar 1, which carries 0.8-kb terminal direct repeats and a spectinomycin resistance determinant, can mediate high-level amplification of an AUD-Sar 1-derived 8.0-kb DNA sequence not only in S. achromogenes but also in the heterologous host Streptomyces lividans. This was seen upon introduction of AUD-Sar 1 into chloramphenicol-sensitive strains of S. lividans via the temperature-sensitive (39 degrees C) plasmid pMT660, which contains the thiostrepton resistance gene tsr. Following the cultivation of transformants at 39 degrees C on media containing spectinomycin, a number of strains which were unable to grow on thiostrepton and which carried the amplified 8.0-kb DNA sequence as arrays of 200 to 300 copies of tandem 8.0-kb repeats were found. Chloramphenicol-resistant strains of S. lividans did not yield amplified sequences under similar conditions. Studies with plasmids carrying inserted antibiotic resistance genes at two sites of AUD-Sar 1 yielded coamplified sequences which contain the inserted DNA. Transformation with a plasmid carrying a 1.0-kb deletion in AUD-Sar 1 followed by growth under similar conditions yielded a 7.0-kb repeated DNA sequence. Southern analysis revealed the absence of vector sequences located on the right side of AUD-Sar 1 in the input plasmids in all examined DNA samples of amplified strains. In contrast, a majority of the samples revealed the presence at unit copy level of AUD-Sar 1 left-adjacent sequences which are part of the input plasmids and in several samples the presence of certain vector sequences located near them. The results suggest input plasmid integration into the S. lividans chromosome prior to the generation of the amplified sequences and the deletion of AUD-Sar 1 adjacent sequences.

DNA amplification in Streptomyces achromogenes subsp. rubradiris is accompanied by a deletion, and the amplified sequences are conditionally stable and can be eliminated by two pathways.

Southern blot analysis of BglII-digested DNA isolated from wild-type Streptomyces achromogenes, which harbors the 8.8-kilobase amplifiable unit of DNA, AUD-Sar 1, and of similarly digested DNA from 12 strains carrying an array of 200 to 300 tandem copies of a specific AUD-Sar 1-derived 8.0-kilobase DNA sequence, ADS-Sar 1, revealed the absence of the 12.4-kilobase BglII AUD-Sar 1-chromosome right junction band in the latter strains, whereas the corresponding 26.0-kilobase left junction band remained unaltered. Further Southern analyses indicated in all of the seven amplified strains tested the occurrence of a deletion of at least 10 kilobases of the DNA adjacent to the right side of the AUD. The deletion has one endpoint in the vicinity of the ADS array. Corroborating and expanding upon previously reported results, we found that the amplified DNA of strain C010 was stably maintained for at least 20 transfers when the transfers involved mycelia propagated in spectinomycin-free liquid medium. In contrast, when strain C010 was subjected separately to one cycle of protoplast formation and regeneration or to three cycles of spore germination, aerial mycelium formation, and sporulation on spectinomycin-free media, only approximately 20% of the protoplast regenerants and spores retained the reiterated DNA sequences and the ability subsequently to form colonies on media containing high levels of spectinomycin. Approximately 80% of these units completely deleted the reiterated DNA and left adjacent sequences and exhibited sensitivity to 25 micrograms of spectinomycin per ml. One among 24 protoplast-derived deletants apparently retained the left portion of the AUD-ADS left direct repeat plus left adjacent sequences.

Spectinomycin resistance and associated DNA amplification in Streptomyces achromogenes subsp. rubradiris.

Streptomyces achromogenes subsp. rubradiris plated at low density on 1,000 micrograms of spectinomycin per ml initially produces slow-growing, bald colonies from which arise, in a spatially and temporally random fashion, foci of rapidly growing aerial mycelium-forming cells whose DNA contains an approximately 200- to 300-fold amplification of an 8-kilobase (kb) sequence. This sequence was cloned in Escherichia coli on pBR322 and physically characterized. It was separately cloned also in Streptomyces lividans as a BglII fragment and shown to impart high-level resistance to spectinomycin in an orientation-independent manner when present in either the high-copy-number vector pIJ702 or the unit-copy-number vector pIJ943. A spectinomycin resistance determinant was shown to reside on a 1.7-kb SphI-BglII subfragment. Analysis of Southern blots of restriction enzyme digests of wild-type S. achromogenes DNA probed with the labeled 8-kb DNA sequence resulted in the identification and subsequent cloning in S. lividans of a 10.4-kb BamHI fragment which probably includes the complete 8.8-kb amplifiable unit of DNA. This unit is present in wild-type S. achromogenes and in the initially slow-growing, bald colonies arising on 1,000 micrograms of spectinomycin per ml as a single copy. It carries two 0.8-kb direct repeats at its termini as well as the spectinomycin resistance determinant close to one of these termini. About 5% of protoplast regenerants from wild-type S. achromogenes and 77% of protoplast regenerants from the rapidly growing strains lost both the ability to grow on spectinomycin at 10 micrograms/ml and the sequences that hybridize with the 8-kb probe DNA. The 1.7-kb Bg/II-SphI resistance fragment, when introduced via the vector pIJ702 into an S. achromogenes strain sensitive to 10 microgram of spectinomycin per ml, permitted its vigorous growth on 1,000 micrograms of the antibiotic per ml.