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.

Regulation of the synthesis of lactate dehydrogenase-X during spermatogenesis in the mouse.

Total mouse testis RNA directs the synthesis of the sperm-specific C subunit of lactate dehydrogenase-X (LDH-X) when translated in a cell-free system derived from rabbit reticulocytes. The newly synthesized C subunits were isolated by immunoprecipitation with antibody specific for this isozyme, and quantitated by electrophoresis on SDS polyacrylamide gels. The amount of radioactivity incorporated into the enzyme subunit was directly proportional to the amount of testis RNA added to the translational system, thereby providing a sensitive and reliable method for assessing relative LDH-X mRNA activity. A combination of sucrose gradient centrifugation and oligo(dT)-cellulose chromatography resulted in a 23-fold purification of LDH-X mRNA over total cytoplasmic testis RNA. Analysis of LDH-X mRNA activity in the developing testis indicated that the appearance of functional LDH-X mRNA activity coincides with the appearance of LDH-X catalytic activity at 14 d postpartum. Measurement of LDH-X mRNA levels in separated testis cell populations prepared by centrifugal elutriation demonstrated that LDH-X mRNA represents 0.17-0.18% of the total functional mRNA activity in fractions enriched in pachytene spermatocytes and round spermatids, but only 0.09-0.10% of the translation products of elongated spermatids.

U1 precursors: variant 3' flanking sequences are transcribed in human cells.

Using RNase protection and oligonucleotide hybridization experiments, we have shown that U1 precursors are derived by transcription of 3' flanking sequences. A labeled SP6 transcript of one of the true U1 genes (pD2) was able to protect a subset of the 3' flanking sequences present in HeLa cytoplasmic U1 RNA. However, not all U1 precursors were protected using this probe, suggesting that variant U1 precursor 3' tail sequences are expressed in HeLa cells. This conclusion has been confirmed by hybridization of HeLa RNA samples with specific oligonucleotide probes representing variant U1 3' flanking sequences. Interestingly, these variant tail sequences contain the putative Sm antigen binding site, A(U)3-6G. The conservation of this flanking sequence through evolution suggests a possible functional role for these precursor tails in ordering protein binding to U1 RNA.

U1 small nuclear ribonucleoprotein studied by in vitro assembly.

The small nuclear RNAs are known to be complexed with proteins in the cell (snRNP). To learn more about these proteins, we developed an in vitro system for studying their interactions with individual small nuclear RNA species. Translation of HeLa cell poly(A)+ mRNA in an exogenous message-dependent reticulocyte lysate results in the synthesis of snRNP proteins. Addition of human small nuclear RNA U1 to the translation products leads to the formation of a U1 RNA-protein complex that is recognized by a human autoimmune antibody specific for U1 snRNP. This antibody does not react with free U1 RNA. Moreover, addition of a 10- to 20-fold molar excess of transfer RNA instead of U1 RNA does not lead to the formation of an antibody-recognized RNP. The proteins forming the specific complex with U1 RNA correspond to the A, B1, and B2 species (32,000, 27,000, and 26,000 mol wt, respectively) observed in previous studies with U1 snRNP obtained by antibody-precipitation of nuclear extracts. The availability of this in vitro system now permits, for the first time, direct analysis of snRNA-protein binding interactions and, in addition, provides useful information on the mRNAs for snRNP proteins.

Intracellular site of U1 small nuclear RNA processing and ribonucleoprotein assembly.

We have investigated the intracellular site and posttranscriptional immediacy of U1 small nuclear RNA processing and ribonucleoprotein (RNP) assembly in HeLa cells. After 30 or 45 min of labeling with [3H]uridine, a large amount of U1-related RNA radioactivity in the cytoplasm was found by using either hypotonic or isotonic homogenization buffers. The pulse-labeled cytoplasmic U1 RNA was resolved as a ladder of closely spaced bands running just behind mature-size U1 (165 nucleotides) on RNA sequencing gels, corresponding to a series of molecules between one and at least eight nucleotides longer than mature U1. They were further identified as U1 RNA sequences by gel blot hybridization with cloned U1 DNA. The ladder of cytoplasmic U1 RNA bands reacted with both RNP and Sm autoimmune sera and with a monoclonal Sm antibody, indicating a cytoplasmic assembly of these U1 RNA-related molecules into complexes containing the same antigens as nuclear U1 RNP particles. The cytoplasmic molecules behave as precursors to mature nuclear U1 RNA in both pulse-chase and continuous labeling experiments. While not excluding earlier or subsequent nuclear stages, these results suggest that the cytoplasm is a site of significant U1 RNA processing and RNP assembly. This raises the possibility that nuclear-transcribed eucaryotic RNAs are always processed in the cell compartment other than that in which they ultimately function, which suggests a set of precise signals regulating RNA and ribonucleoprotein traffic between nucleus and cytoplasm.

Precursors of U4 small nuclear RNA.

The processing and ribonucleoprotein assembly of U4 small nuclear RNA has been investigated in HeLa cells. After a 45-min pulse label with [3H]uridine, a set of apparently cytoplasmic RNAs was observed migrating just behind the gel electrophoretic position of mature U4 RNA. These molecules were estimated to be one to at least seven nucleotides longer than mature U4 RNA. They reacted with Sm autoimmune patient sera and a monoclonal Sm antibody, indicating their association with proteins characteristic of small nuclear ribonucleoprotein complexes. The same set of RNAs was identified by hybrid selection of pulse-labeled RNA with cloned U4 DNA, confirming that these are U4 RNA sequences. No larger nuclear precursors of these RNAs were detected. Pulse-chase experiments revealed a progressive decrease in the radioactivity of the U4 precursor RNAs coincident with an accumulation of labeled mature U4 RNA, confirming a precursor-product relationship.

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.

Small nuclear ribonucleoproteins of Drosophila: identification of U1 RNA-associated proteins and their behavior during heat shock.

In Drosophila, two nuclear proteins of approximately 26,000 and 14,000 molecular weight are recognized by a human autoimmune antibody for mammalian ribonucleoprotein (RNP) particles that contain U1 small nuclear RNA. The antibody-selected Drosophila RNP contains, in addition to these two proteins, a single RNA species that has been identified as U1 by hybridization with a cloned Drosophila U1 DNA probe. Small nuclear RNP isolated from human cells under the same conditions as used for Drosophila and selected by the anti-U1 RNP-specific antibody contains eight proteins, two of which are similar in molecular weight to the two Drosophila U1 RNP proteins. Thus, even though the nucleotide sequences of Drosophila and human U1 RNA are about 72% homologous, and the corresponding RNPs are both recognized by the same human autoantibody, Drosophila U1 RNP appears to have a simpler protein complement than its mammalian counterpart. The two Drosophila U1 RNA-associated proteins are synthesized at normal or slightly increased rates during the heat shock response and are incorporated into antibody-recognizable RNP complexes. This raises the possibility that U1 RNP is an indispensable nuclear element for cell survival during heat shock.

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.

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.

Ribonucleoprotein organization of eukaryotic RNA. XXXII. U2 small nuclear RNA precursors and their accurate 3' processing in vitro as ribonucleoprotein particles.

Biosynthetic precursors of U2 small nuclear RNA have been identified in cultured human cells by hybrid-selection of pulse-labeled RNA with cloned U2 DNA. These precursor molecules are one to approximately 16 nucleotides longer than mature U2 RNA and contain 2,2,7-trimethylguanosine "caps". The U2 RNA precursors are associated with proteins that react with a monoclonal antibody for antigens characteristic of small nuclear ribonucleoprotein particles. Like previously described precursors of U1 and U4 small nuclear RNAs, the pre-U2 RNAs are recovered in cytoplasmic fractions, although it is not known if this is their location in vivo. The precursors are processed to mature-size U2 RNA when cytoplasmic extracts are incubated in vitro at 37 degrees C. Mg2+ is required but ATP is not. The ribonucleoprotein structure of the pre-U2 RNA is maintained during the processing reaction in vitro, as are the 2,2,7-trimethylguanosine caps. The ribonucleoprotein organization is of major importance, as exogenous, protein-free U2 RNA precursors are degraded rapidly in the in vitro system. Two lines of evidence indicate that the conversion of U2 precursors to mature-size U2 RNA involves a 3' processing reaction. First, the reaction is unaffected by a large excess of mature U2 small nuclear RNP, whose 5' trimethylguanosine caps would be expected to compete for a 5' processing activity. Second, when pre-U2 RNA precursors are first stoichiometrically decorated with an antibody specific for 2,2,7-trimethylguanosine, the extent of subsequent processing in vitro is unaffected. These results provide the first demonstration of a eukaryotic RNA processing reaction in vitro occurring within a ribonucleoprotein particle.

The small nuclear RNAs of Drosophila.

We have investigated the sequences of the major small nuclear RNAs of Drosophila cultured cells, with the objective of elucidating phylogenetically conserved primary and secondary structures by comparison of the data with previously determined sequences of these RNAs in vertebrate species. Our results reveal striking degrees of conservation between each Drosophila RNA and its vertebrate cognate, and also demonstrate blocks of homology among the Drosophila small nuclear RNAs, as previously described for vertebrates. The most conserved features include the 5' terminal region of U1 RNA, though to function in pre-mRNA splicing, most of the regions of U4 RNA recently implicated in 3' processing of pre-mRNA, and the major snRNP protein binding site ("domain A") that is also shared by vertebrate U1, U2, U4 and U5 RNAs. Several other conserved features have been revealed, suggesting additional regions of functional significance in these RNAs and also providing further insights into the evolutionary history of the small nuclear RNAs.

Androgens affect the processing of secretory protein precursors in the guinea pig seminal vesicle. I. Evidence for androgen-regulated proteolytic processing.

The guinea pig seminal vesicle epithelium synthesizes and secretes four major secretory proteins (SVP-1-4). Previous work has established that these four proteins are cleaved from two primary translation products in a complex series of protein processing reactions. The present studies suggest that these protein processing reactions are regulated by androgens. In vitro labeling of seminal vesicle proteins revealed significant differences in the patterns of secretory protein intermediates produced by tissue from intact and castrated animals. Seminal vesicle tissue explants from castrated animals secreted a subset of the processing intermediates secreted by tissue from intact animals. The changes in the patterns of secretory protein intermediates became more pronounced with increasing time after castration, and were fully reversible by treatment of castrated animals with testosterone, suggesting that androgens were affecting the processing or secretion of secretory protein precursors. Amino-terminal protein sequencing of secretory protein processing intermediates that accumulate in the seminal vesicle lumen after castration suggests that the guinea pig seminal vesicle contains an androgen-regulated proteolytic processing activity.

Androgens affect the processing of secretory protein precursors in the guinea pig seminal vesicle. II. Identification of conserved sites for protein processing.

The guinea pig seminal vesicle epithelium is an androgen-dependent tissue that synthesizes and secretes four major secretory proteins (SVP-1, SVP-2, SVP-3, and SVP-4). Sequencing of near full-length cDNA clones corresponding to the two most abundant mRNAs produced by the seminal vesicle reveals that all four secretory proteins are cleaved from two secretory protein precursors. Amino acid sequences from purified SVP-2 match the central region of the predicted amino acid sequences from the smaller cDNA clone, GP2 (581 nucleotides). Similar analysis demonstrates that the predicted amino acid sequence from the longer cDNA clone, GP1 (1368 nucleotides), codes for the related proteins SVP-3 and SVP-4 as well as SVP-1. The 43.2 kilodalton polyprotein precursor coded by GP1 contains two different sets of 24 amino acid tandemly repeated sequences. The two secretory protein precursors have extensive regions of peptide sequence homology, particularly in regions where protein processing must occur to produce the mature secretory proteins. Analysis of the predicted secondary structure of the two precursor polypeptides revealed a strong correlation between structural features and sites of protein processing.

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.

The effects of androgen on the transcription of specific genes in guinea pig seminal vesicle epithelium.

Steroid hormones have been shown to have highly differential effects on the expression of abundant cell-specific protein genes in a multitude of model tissues. In rat seminal vesicle, for example, DNA clones representing two major secretory protein genes have been used to show that both of the genes are differentially regulated by androgen. In this paper, we have examined the effects of androgen on the transcription of two major secretory protein genes in guinea pig seminal vesicle epithelium. Nuclear run-off experiments were used to show that castration of the adult resulted in a 3-fold decrease in total transcription activity. Surprisingly, the decrease in total transcriptional activity was not reflected in a differential decrease in the transcriptional activity of the two major secretory protein genes. When the effects of castration on the transcriptional activity of the major secretory protein genes were compared to the effects on other genes, it was found that the transcriptional activity of each gene examined was decreased by the same magnitude as the major secretory protein genes. Similarly, the transcriptional activity of every gene examined increased by the same magnitude as the major secretory protein genes during hormone repletion of the castrated adult. Thus, in contrast to the differential effects of steroids on the transcription of abundant cell-specific proteins in many other steroid-dependent tissues, the transcription of major secretory proteins in guinea pig seminal vesicle epithelium appears to be regulated in parallel with many other genes. The generalized effects of androgen on transcriptional activity could account for the generalized effects of androgen on seminal vesicle epithelial cell structure and function.

Molecular diversity of neuronal-type calcium channels identified in small cell lung carcinoma.

Using the polymerase chain reaction (PCR), we identified RNA transcripts for two distinct classes of neuronal-type voltage-gated Ca2+ channels (VGCC) in a prototypic small cell lung carcinoma (SCLC) cell line, SCC-9. Oligonucleotide primers were designed to encode amino acid sequences common to alpha 1-subunits of known neuronal VGCC classes. Sequencing of complementary DNA (cDNA) clones derived from two independent PCR products revealed that one corresponded to a brain class A VGCC fragment predicted to encode a P-type VGCC (insensitive to dihydropyridines and omega-conotoxin) characteristic of cerebellar Purkinje cells but not previously identified in humans. The second PCR product was identical (except for one conservative nucleotide difference) to a fragment of the class D VGCC of neurons and neuroendocrine cells, which encodes an L-type VGCC (sensitive to dihydropyridines). By Northern blot analyses, both cDNAs hybridized to messenger RNAs (mRNAs) obtained from SCC-9; class D hybridized additionally to human cerebral cortical mRNA, but neither hybridized to mRNA from the skeletal muscle cell line TE671. Although no cDNA corresponding to class B VGCC (N-type) was identified, SCLCs are known to express VGCC that are sensitive to omega-conotoxin and coprecipitate with 125I-labeled-omega-conotoxin when complexed with serum IgG from patients with the Lambert-Eaton myasthenic syndrome. The multiple classes of neuronal-type VGCC expressed in SCLC could conceivably have both unique and related antigenic determinants that may give rise to antineuronal autoimmune responses. This would account for a spectrum of paraneoplastic neurologic disorders including the Lambert-Eaton syndrome and subacute cerebellar degeneration.

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.

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.

The snRNP E protein multigene family contains five pseudogenes with common mutations.

Sequence data from three previously-uncharacterized members of the snRNP E protein multigene family suggest that each is a non-transcribed processed pseudogene, even though one clone has the potential to code for a full-length protein with greater than 90% similarity to previously-characterized E protein cDNAs. Each of the newly-analyzed family members is without introns, contains a tract of polyadenylic acid residues, and is flanked by short direct repeats. In addition, the three sequences all contain point mutations that distinguish them from the E protein coding sequence. Seven point mutations are common to the three sequences described here and to two previously-described E protein pseudogenes. Although all of these mutations are transitions, only 5 of 7 could have been generated by deamination of methylated cytosines in inactive genes. Thus, the common mutations in the pseudogenes suggest an origin other than the expressed gene that we have described. Allelic variants for two of the pseudogenes were detected and repetitive elements are located near four of the five E protein pseudogenes that have been characterized.