A prospective genome-wide study of prostate cancer metastases reveals association of wnt pathway activation and increased cell cycle proliferation with primary resistance to abiraterone acetate-prednisone.

Background: Genomic aberrations have been identified in metastatic castration-resistant prostate cancer (mCRPC), but molecular predictors of resistance to abiraterone acetate/prednisone (AA/P) treatment are not known. Patients and methods: In a prospective clinical trial, mCRPC patients underwent whole-exome sequencing (n = 82) and RNA sequencing (n = 75) of metastatic biopsies before initiating AA/P with the objective of identifying genomic alterations associated with resistance to AA/P. Primary resistance was determined at 12 weeks of treatment using criteria for progression that included serum prostate-specific antigen measurement, bone and computerized tomography imaging and symptom assessments. Acquired resistance was determined using the end point of time to treatment change (TTTC), defined as time from enrollment until change in treatment from progressive disease. Associations of genomic and transcriptomic alterations with primary resistance were determined using logistic regression, Fisher's exact test, single and multivariate analyses. Cox regression models were utilized for determining association of genomic and transcriptomic alterations with TTTC. Results: At 12 weeks, 32 patients in the cohort had progressed (nonresponders). Median study follow-up was 32.1 months by which time 58 patients had switched treatments due to progression. Median TTTC was 10.1 months (interquartile range: 4.4-24.1). Genes in the Wnt/ß-catenin pathway were more frequently mutated and negative regulators of Wnt/ß-catenin signaling were more frequently deleted or displayed reduced mRNA expression in nonresponders. Additionally, mRNA expression of cell cycle regulatory genes was increased in nonresponders. In multivariate models, increased cell cycle proliferation scores (≥ 50) were associated with shorter TTTC (hazard ratio = 2.11, 95% confidence interval: 1.17-3.80; P = 0.01). Conclusions: Wnt/ß-catenin pathway activation and increased cell cycle progression scores can serve as molecular markers for predicting resistance to AA/P therapy.

Pharmacogenetics of the mycophenolic acid targets inosine monophosphate dehydrogenases IMPDH1 and IMPDH2: gene sequence variation and functional genomics.

BACKGROUND AND PURPOSE: Inosine monophosphate dehydrogenases, encoded by IMPDH1 and IMPDH2, are targets for the important immunosuppressive drug, mycophenolic acid (MPA). Variation in MPA response may result, in part, from genetic variation in IMPDH1 and IMPDH2. EXPERIMENTAL APPROACH: We resequenced IMPDH1 and IMPDH2 using DNA from 288 individuals from three ethnic groups and performed functional genomic studies of the sequence variants observed. KEY RESULTS: We identified 73 single nucleotide polymorphisms (SNPs) in IMPDH1, 59 novel, and 25 SNPs, 24 novel, in IMPDH2. One novel IMPDH1 allozyme (Leu275) had 10.2% of the wild-type activity as a result of accelerated protein degradation. Decreased activity of the previously reported IMPDH2 Phe263 allozyme was primarily due to decreased protein quantity, also with accelerated degradation. These observations with regard to the functional implications of variant allozymes were supported by the IMPDH1 and IMPDH2 X-ray crystal structures. A novel IMPDH2 intron 1 SNP, G > C IVS1(93), was associated with decreased mRNA quantity, possibly because of altered transcription. CONCLUSIONS AND IMPLICATIONS: These results provide insight into the nature and extent of sequence variation in the IMPDH1 and IMPDH2 genes. They also describe the influence of gene sequence variation that alters the encoded amino acids on IMPDH function and provide a foundation for future translational studies designed to correlate sequence variation in these genes with outcomes in patients treated with MPA.

SLC6A4 variation and citalopram response.

The influence of genetic variations in SLC6A4 (serotonin transporter gene) on citalopram treatment of depression using the Sequenced Treatment to Relieve Depression (STAR*D) sample was assessed. Of primary interest were three previously studied polymorphisms: 1) the VNTR variation of the second intron, 2) the indel promoter polymorphism (5HTTLPR or SERT), and 3) a single nucleotide polymorphism (SNP) rs25531. Additionally, SLC6A4 was resequenced to identify new SNPs for exploratory analyses. DNA from 1914 subjects in the STAR*D study were genotyped for the intron 2 VNTR region, the indel promoter polymorphism, and rs25531. Associations of these variants with remission of depressive symptoms were evaluated following citalopram treatment. In white non-Hispanic subjects, variations in the intron 2 VNTR (point-wise P = 0.041) and the indel promoter polymorphism (point-wise P = 0.039) were associated with remission following treatment with citalopram. The haplotype composed of the three candidate loci was also associated with remission, with a global p-value of 0.040 and a maximum statistic simulation p-value of 0.0031 for the S-a-12 haplotype, under a dominant model. One SNP identified through re-sequencing the SLC6A4 gene, Intron7-83-TC, showed point-wise evidence of association, which did not remain significant after correction for the number of SNPs evaluated in this exploratory analysis. No associations between these SLC6A4 variations and remission were found in the white Hispanic or black subjects. These findings suggest that multiple variations in the SLC6A4 gene are associated with remission in white non-Hispanic depressed adults treated with citalopram. The mechanism of action of these variants remains to be determined.

Genetic diversity and function in the human cytosolic sulfotransferases.

Amino-acid substitutions, which result from common nonsynonymous (NS) polymorphisms, may dramatically alter the function of the encoded protein. Gaining insight into how these substitutions alter function is a step toward acquiring predictability. In this study, we incorporated gene resequencing, functional genomics, amino-acid characterization and crystal structure analysis for the cytosolic sulfotransferases (SULTs) to attempt to gain predictability regarding the function of variant allozymes. Previously, four SULT genes were resequenced in 118 DNA samples. With additional resequencing of the remaining eight SULT family members in the same DNA samples, a total of 217 polymorphisms were revealed. Of 64 polymorphisms identified within 8785 bp of coding regions from SULT genes examined, 25 were synonymous and 39 were NS. Overall, the proportion of synonymous changes was greater than expected from a random distribution of mutations, suggesting the presence of a selective pressure against amino-acid substitutions. Functional data for common variants of five SULT genes have been previously published. These data, together with the SULT1A1 variant allozyme data presented in this paper, showed that the major mechanism by which amino acid changes altered function in a transient expression system was through decreases in immunoreactive protein rather than changes in enzyme kinetics. Additional insight with regard to mechanisms by which NS single nucleotide polymorphisms alter function was sought by analysis of evolutionary conservation, physicochemical properties of the amino-acid substitutions and crystal structure analysis. Neither individual amino-acid characteristics nor structural models were able to accurately and reliably predict the function of variant allozymes. These results suggest that common amino-acid substitutions may not dramatically alter the protein structure, but affect interactions with the cellular environment that are currently not well understood.

Human catechol O-methyltransferase genetic variation: gene resequencing and functional characterization of variant allozymes.

Catechol O-methyltransferase (COMT) plays an important role in the metabolism of catecholamines, catecholestrogens and catechol drugs. A common COMT G472A genetic polymorphism (Val108/158Met) that was identified previously is associated with decreased levels of enzyme activity and has been implicated as a possible risk factor for neuropsychiatric disease. We set out to 'resequence' the human COMT gene using DNA samples from 60 African-American and 60 Caucasian-American subjects. A total of 23 single nucleotide polymorphisms (SNPs), including a novel nonsynonymous cSNP present only in DNA from African-American subjects, and one insertion/deletion were observed. The wild type (WT) and two variant allozymes, Thr52 and Met108, were transiently expressed in COS-1 and HEK293 cells. There was no significant change in level of COMT activity for the Thr52 variant allozyme, but there was a 40% decrease in the level of activity in cells transfected with the Met108 construct. Apparent K(m) values of the WT and variant allozymes for the two reaction cosubstrates differed slightly, but significantly, for 3,4-dihydroxybenzoic acid but not for S-adenosyl-L-methionine. The Met108 allozyme displayed a 70-90% decrease in immunoreactive protein when compared with WT, but there was no significant change in the level of immunoreactive protein for Thr52. A significant decrease in the level of immunoreactive protein was also observed in hepatic biopsy samples from patients homozygous for the allele encoding Met108. These observations represent steps toward an understanding of molecular genetic mechanisms responsible for variation in COMT level and/or properties, variation that may contribute to the pathophysiology of neuropsychiatric disease.

Human cytosolic sulfotransferase database mining: identification of seven novel genes and pseudogenes.

A total of 10 SULT genes are presently known to be expressed in human tissues. We performed a comprehensive genome-wide search for novel SULT genes using two different but complementary approaches, and developed a novel graphical display to aid in the annotation of the hits. Seven novel human SULT genes were identified, five of which were predicted to be pseudogenes, including two processed pseudogenes and three pseudogenes that contained introns. Those five pseudogenes represent the first unambiguous SULT pseudogenes described in any species. Expression-profiling studies were conducted for one novel gene, SULT6B1, and a series of alternatively spliced transcripts were identified in the human testis. SULT6B1 was also present in chimpanzee and gorilla, differing at only seven encoded amino-acid residues among the three species. The results of these database mining studies will aid in studies of the regulation of these SULT genes, provide insights into the evolution of this gene family in humans, and serve as a starting point for comparative genomic studies of SULT genes.

Human sulfotransferase SULT2A1 pharmacogenetics: genotype-to-phenotype studies.

SULT2A1 catalyzes the sulfate conjugation of dehydroepiandrosterone (DHEA) as well as other steroids. As a step toward pharmacogenetic studies, we have 'resequenced' SULT2A1 using 60 DNA samples from African-American and 60 samples from Caucasian-American subjects. All exons, splice junctions and approximately 370 bp located 5' of the site of transcription initiation were sequenced. We observed 15 single nucleotide polymorphisms (SNPs), including three non-synonymous coding SNPs (cSNPs) that were present only in DNA from African-American subjects. Linkage analysis revealed that two of the nonsynonymous cSNPs were tightly linked. Expression constructs were created for all nonsynonymous cSNPs observed, including a 'double variant' construct that included the two linked cSNPs, and those constructs were expressed in COS-1 cells. SULT2A1 activity was significantly decreased for three of the four variant allozymes. Western blot analysis demonstrated that decreased levels of immunoreactive protein appeared to be the major mechanism responsible for decreases in activity, although apparent Km values also varied among the recombinant allozymes. In addition, the most common of the nonsynonymous cSNPs disrupted the portion of SULT2A1 involved with dimerization, and this variant allozyme behaved as a monomer rather than a dimer during gel filtration chromatography. These observations indicate that common genetic polymorphisms for SULT2A1 can result in reductions in levels of both activity and enzyme protein. They also raise the possibility of ethnic-specific pharmacogenetic variation in SULT2A1-catalyzed sulfation of both endogenous and exogenous substrates for this phase II drug-metabolizing enzyme.

Human sulfotransferase SULT1C1 pharmacogenetics: gene resequencing and functional genomic studies.

Sulfotransferase (SULT) enzymes catalyze an important phase II reaction in the biotransformation of many drugs and other xenobiotics. We previously cloned the human SULT1C1 cDNA and gene as steps toward pharmacogenetic studies. We have now 'resequenced' the exons, portions of introns flanking exons and approximately 315 bp of the 5' flanking region of SULT1C1 in 89 DNA samples from Caucasian subjects to identify common genetic polymorphisms. Nineteen separate polymorphisms were observed, including four nonsynonymous coding region single nucleotide polymorphisms (cSNPs) and five insertions/deletions. These data were also used to determine and/or infer common SULT1C1 haplotypes. Three of the four nonsynonymous cSNPs had allele frequencies greater than 1%, including one with a frequency of 6.7%. Expression constructs were created for all of the nonsynonymous cSNPs observed, and those constructs were used to transfect COS-1 cells. Three of the four SULT1C1 variant allozymes had significantly reduced enzyme activity when compared with the wild-type enzyme. Among the variant allozymes, apparent Km values for 3'-phosphoadenosine 5'-phosphosulfate (PAPS), the sulfate donor for the reaction, varied 7-fold, and quantitative Western blot analysis showed variable levels of immunoreactive protein when compared to the wild-type enzyme. Therefore, mechanisms responsible for decreased activity involved both alterations in levels of enzyme protein and alterations in substrate kinetics. In summary, application of a 'genotype to phenotype' strategy has resulted in the identification of a series of functionally significant common genetic polymorphisms for SULT1C1. It will now be possible to evaluate the possible contribution of these polymorphisms to variation in the sulfate conjugation of drugs, other xenobiotics and/or disease pathophysiology.

Cyclin-dependent kinase 5 is expressed in both Sertoli cells and metaphase spermatocytes.

OBJECTIVE: To gain insight into the function of cyclin-dependent kinase 5 (Cdk5) in spermatogenesis. DESIGN: The expression of the Cdk5 protein was determined with the use of immunohistochemical and immunoblot analysis. SETTING: Academic research laboratory. ANIMAL(S): Adult mouse and archival human testicular tissue were used for the immunohistochemical analysis. Adult mice were used as the source of tissues for the immunoblot analysis. INTERVENTION(S): The immunohistochemical analysis was performed with an anti-Cdk5 antibody. The double immunohistochemical analysis was performed with anti-Cdk5 and alpha-tubulin antibodies. Immunoblotting was used to examine multiple mouse tissues for Cdk5 expression. MAIN OUTCOME MEASURE(S): Analysis of Cdk5 protein distribution. RESULT(S): Cdk5 was localized specifically within the cytoplasm of Sertoli cells and meiotic metaphase germ cells. The double immunohistochemistry analysis demonstrated the co-localization of Cdk5 and alpha-tubulin within the Sertoli cells. Western blot analysis revealed a high level of expression of Cdk5 in the testicular lysate. CONCLUSION(S): The cyclin-dependent kinases are known regulators of the cell cycle; however, Cdk5 expression previously has been described in terminally differentiated cells of the brain. The present evidence of an association between Cdk5 and microfilaments of Sertoli cells and meiotic metaphase germ cells suggests a role of Cdk5 in both seminiferous tubule function and meiosis.

In vivo expression of a variant human U6 RNA from a unique, internal promoter.

We previously isolated a variant of the human U6 small nuclear RNA gene (87U6) and demonstrated that transcription of this gene is controlled by a novel internal promoter. It has now been shown that two blocks of sequence within the coding region are both necessary and sufficient to direct expression of 87U6 in transcription assays performed in vitro. In addition, 87U6 is expressed in vivo and can assemble into snRNP complexes. Specific primer extension assays on total RNA from HeLa cells shows that 87U6 RNA is present in these cells. Also, microinjection of plasmid encoded 87U6 genes into Xenopus laevis oocyte nuclei results in the expression of this variant RNA. Immunoprecipitation with anti-Sm antibodies suggests that 87U6 RNA assembles into a snRNP particle with U4 snRNA. Finally, the variant snRNA is capped with the U6 specific gamma-monomethyl phosphate cap when incubated in HeLa extracts. These data suggest that 87U6 RNA may function in the splicing process, in a manner similar to the wild-type U6 RNA. The recent observations of a minor class of mRNA introns that are spliced by a distinct collection of snRNP particles suggest an important role for variant snRNAs in the splicing of transcripts with alternative splice junctions.

TGFbeta-inducible early gene (TIEG) also codes for early growth response alpha (EGRalpha): evidence of multiple transcripts from alternate promoters.

TGFbeta-inducible early gene (TIEG) and early growth response alpha (EGRalpha) are putative transcription factors based on homology to known zinc finger proteins SP1, EGR1, BTEB, and Wilm tumor. Here we report that TIEG and EGRalpha are expressed from alternative promoters of the same gene. The TIEG/EGRalpha gene spans 8 kb and contains five exons. Use of alternative first exons results in TIEG having 12 unique amino acids on its N-terminus. Computer analysis of the 5' upstream regions of either TIEG (exon 1a) or EGRalpha (exon 1b) does not identify a TATA box or initiator sequencebut shows consensus sequence similarities to binding sites for several transcription factors including SP1,JunB, and aromatic hydrocarbon/receptor-ligand complexes. Analysis of constructs containing 5'-flanking regions show that both the TIEG and the EGRalpha promoters have significant activity in human fetal osteoblast cells. Northern analysis of mRNA from various human tissues and several cell lines reveals that TIEG is the predominant transcript produced and regulated by growth factors from the TIEG/EGRalpha gene.

Production of SVP-1/-3/-4 in guinea pig testis. Characterization of novel transcripts containing long 5'-untranslated regions and multiple upstream AUG codons.

The GP1G gene of the guinea pig codes for three of the four abundant seminal vesicle secretory proteins produced in this species. This gene is expressed at highest efficiency in the seminal vesicle (SV) from a promoter that contains a canonical TATA box and CCAAT box. However, GP1G gene transcripts and proteins have also been identified in other tissues. To investigate the structure of GP1G transcripts produced in the testis, cDNA clones were isolated by screening a testis library. Three unique cDNAs (TSM1-3) were isolated. Each of these clones contained a 3'-untranslated region (UTR) and coding region identical to that of the seminal vesicle transcript. However, the 5'-UTRs of the testis transcripts were significantly longer than that found on the SV mRNA (416-646 nucleotides compared with only 23 nucleotides for the SV). Each of these alternatively spliced 5'-UTRs incorporated the SV promoter elements into transcribed sequence, and each contained multiple upstream AUG codons predicted to abolish translation of the major open reading frame. Nevertheless, each of the testis transcripts was capable of directing the synthesis of GP1G-related proteins in vitro. Analysis of the translation products suggests that the extended 5'-UTR of the testis transcripts regulate both the choice of translation start site and the efficiency of translation in this system. Western blot analysis of testis proteins revealed that the protein products of GP1G are also synthesized by the testis in vivo.

Structure of the rat collagen IV promoter.

We have isolated a 1.6 kb clone from a rat genomic library which contains the bidirectional collagen IV promoter, flanked by exons coding for the alpha 1 (IV) and alpha 2 (IV) collagen chains. There are at least two transcription start sites within both the alpha 1 (IV) and alpha 2 (IV) collagen genes. Rat mesangial cells were transfected with chloramphenicol acetyltransferase (CAT) reporter plasmids containing segments of the promoter and 5' flanking region, in both the alpha 1 (IV) and alpha 2 (IV) orientations. Our results suggest that transcriptional efficiency of the bidirectional promoter is more efficient in the alpha 2 (IV) direction than in the alpha 1 (IV) direction.

Exons lost and found. Unusual evolution of a seminal vesicle transglutaminase substrate.

The GP1G gene codes for three of the four abundant androgen-regulated secretory proteins produced by the guinea pig seminal vesicle. Sequencing of the entire 6.3-kilobase gene and comparison with other mammalian seminal vesicle secretory protein genes reveals a common three-exon, two-intron organization. However, significant sequence similarity between this group of genes is largely limited to their 5'-flanking regions and first exons, which code almost exclusively for signal peptides in each case. The first intron of GP1G does contain a region with high similarity to the coding exon of a human seminal vesicle secretory protein gene, semenogelin II. The 3' half of the GP1G gene appears to share a common ancestry with the human SKALP/elafin gene. Sequences related to the elafin promoter, coding, untranslated regions, and introns are clearly identifiable within the GP1G sequence. The elafin gene codes for a serine protease inhibitor and is expressed in a variety of different human tissues. To determine if the GP1G gene was also active outside of the seminal vesicle, RNA from a variety of guinea pig tissues was hybridized to a GP1G cDNA probe. At least three novel RNA bands hybridizing to the GP1G probe were detected in testis RNA samples, and GP1G-related mRNAs were also found in other tissues. These data suggest that these seminal vesicle secretory proteins may have functional roles outside the reproductive system.

Abnormal processing of the human cholecystokinin receptor gene in association with gallstones and obesity.

BACKGROUND & AIMS: Cholesterol gallstone disease and obesity are often associated and share the potential, yet unreported, common etiology of cholecystokinin (CCK) dysfunction. While cloning the human CCK-A receptor complementary DNA (cDNA), we found predominance of a 262-base pair coding region deletion in a cDNA library prepared from a patient with this phenotype. The aim of this study was to determine the abundance, functional significance, and mechanism for generating this gene product. METHODS: Relative abundance of CCK receptor gene products was determined using polymerase chain reaction and hybridization analysis. Constructs were expressed in COS cells and studied for radioligand binding and intracellular calcium responses. A human genomic clone for this receptor was sequenced, and the critical regions were compared with those of the patient. RESULTS: Ninety-three percent of the patient's CCK receptor transcripts contained the 262-base pair deletion, whereas only 1.5% +/- 0.9% of control patients had the deletion. This encoded a receptor that did not bind or signal. The deletion corresponded with the third exon; however, this sequence and flanking introns were normal in the patient. CONCLUSIONS: Abnormality of processing an apparently normal CCK receptor gene yields the predominant product with an absent third exon and encoding a nonfunctional receptor, probably reflecting a defective trans-acting splicing factor. An atypical lariat region in the third intron may explain the presence of small amounts of this product in control patients.

Inhibitors of renal epithelial phosphate transport in tumor-induced osteomalacia and uremia.

Tumors such as sclerosing hemangiomas are sometimes associated with hypophosphatemia and osteomalacia, both of which disappear on removal of the tumor. We identified a heat labile, 8,000-25,000 dalton, inhibitor of renal epithelial phosphate transport in supernatants of cultured sclerosing hemangioma cells obtained from a patient with oncogenic osteomalacia and hypophosphatemia. The inhibitor does not alter glucose or alanine transport in renal epithelial cells, and has a mechanism of cellular action distinct from that of parathyroid hormone (PTH) in that it inhibits phosphate transport in renal epithelia without increasing concentrations of cyclic 3',5' adenosine monophosphate (cAMP); it's activity is not blocked by a PTH receptor antagonist. Sclerosing hemangioma cells also produce a material that cross-reacts with antisera directed against PTH and tumor tissue sections immunostain with PTH antibodies. We have characterized a cDNA that encodes the PTH immunoreactive material. In its longest open reading frame the cDNA encodes a protein of 381 amino acids that does not resemble PTH in its primary structure. Opossum kidney cells transfected with the cDNA do not produce a product that inhibits phosphate transport. Dialysates from patients with end-stage renal disease also contain a substance(s) that inhibits phosphate and glucose transport in opossum kidney cells. The inhibitor(s) of phosphate uptake in dialysates is a heat labile, approximately 30,000 dalton substance that inhibits phosphate transport by a cAMP-independent mechanism. Determination of the structures and physiology of these phosphate transport inhibitors is likely to yield insights into the control of phosphate homeostasis.

Human dehydroepiandrosterone sulfotransferase gene: molecular cloning and structural characterization.

Dehydroepiandrosterone sulfotransferase (DHEA ST) catalyzes the sulfate conjugation of DHEA and other steroids. From 20 to 25% of subjects are included in a subgroup with high levels of hepatic DHEA ST activity, raising the possibility that this enzyme activity might be controlled by a genetic polymorphism. To understand the molecular mechanisms involved in regulating levels of DHEA ST activity in human tissue, we cloned the human DHEA ST gene, STD. STD spans at least 17 kb and is composed of 6 exons and 5 introns. The locations of the splice junctions for several of the introns are identical to those present in the rat phenol or aryl ST gene, the only other cytosolic ST gene for which the entire exon/intron structure has been reported, as well as those present in two partially characterized genes for the rat senescence marker protein, genes that are also thought to encode ST enzymes. The 5'-flanking region of the human STD gene does not contain canonical TATA or CCAAT elements, but this region is capable of promoting transcription of a reporter gene in Hep G2 cells. Molecular cloning and structural characterization of the human STD gene will make it possible to study genetic mechanisms involved in the regulation of DHEA ST activity in human tissue.

Thiopurine methyltransferase pharmacogenetics. Cloning of human liver cDNA and a processed pseudogene on human chromosome 18q21.1.

Thiopurine methyltransferase (TPMT) is a genetically polymorphic enzyme that catalyzes the S-methylation of thiopurine drugs such as 6-mercaptopurine. This genetic polymorphism is an important factor responsible for individual variation in thiopurine drug response. A cDNA for TPMT has been cloned from T84 human colon carcinoma cells. Northern blot analysis of multiple human tissues was performed with the T84 human colon carcinoma TPMT cDNA open reading frame (ORF) as a probe as one step toward understanding the molecular basis for the TPMT genetic polymorphism. Three mRNA species (approximately 1.4, 2.0, and 3.6 kb in length) were present in all tissues studied, including liver. However, none of these mRNAs matched the length of the 2.7 kb T84 TPMT cDNA. Therefore, it was important to clone a TPMT cDNA from a human drug-metabolizing organ such as the liver to determine whether its sequence matched that of the cDNA cloned from the T84 cell line. A human liver cDNA library was screened with the T84 TPMT cDNA ORF as a probe, and a 1.8 kb cDNA was isolated with a coding region sequence identical to that of the T84 TPMT cDNA. The TPMT cDNA ORF was then used to screen a human lymphocytes genomic DNA library in an attempt to clone the TPMT gene(s) in humans. Three intronless clones were isolated with identical ORF sequences that were 96% identical to that of the TPMT cDNA, but which contained multiple nucleotide substitutions and one deletion. The 3'- and 5'-flanking regions of one of the genomic DNA clones were sequenced.(ABSTRACT TRUNCATED AT 250 WORDS)

Transcription of a variant human U6 small nuclear RNA gene is controlled by a novel, internal RNA polymerase III promoter.

Promoter elements in the 5' flanking regions of vertebrate U6 RNA genes have been shown to be both necessary and sufficient for transcription by RNA polymerase III. We have recently isolated and characterized a variant human U6 gene (87U6) that can be transcribed by RNA polymerase III in vitro in the absence of any natural 5' or 3' flanking sequences. This gene contains 10 nucleotide differences from the previously characterized human U6 gene (wtU6) within the coding region but has no homology to wtU6 in the upstream promoter region. By constructing chimeras between these two genes, we have shown that mutation of as few as two nucleotides in the coding region of the human U6 RNA gene is sufficient to create an internal promoter that is functional in vitro. A T-to-C transition at position 57 and a single T deletion at position 52 produce an internal U6 promoter that is nearly as active in vitro as the external U6 polymerase III promoter utilized by wtU6. Neither of these residues is absolutely conserved during evolution, and both of these nucleotide changes occur within the previously noted A box homology. Deletion and linker scanning mutations within the coding region of this variant U6 gene suggest that, in addition to the central region including bp 52 and 57, sequences at the extreme 5' end of the gene are critical for efficient transcription. In contrast, flanking sequences have a minor effect on transcriptional efficiency. This arrangement is unique among internal RNA polymerase III promoters and may indicate unique regulation of this gene.