The gene mutated in bare patches and striated mice encodes a novel 3beta-hydroxysteroid dehydrogenase.

X-linked dominant disorders that are exclusively lethal prenatally in hemizygous males have been described in human and mouse. None of the genes responsible has been isolated in either species. The bare patches (Bpa) and striated (Str) mouse mutations were originally identified in female offspring of X-irradiated males. Subsequently, additional independent alleles were described. We have previously mapped these X-linked dominant, male-lethal mutations to an overlapping region of 600 kb that is homologous to human Xq28 (ref. 4) and identified several candidate genes in this interval. Here we report mutations in one of these genes, Nsdhl, encoding an NAD(P)H steroid dehydrogenase-like protein, in two independent Bpa and three independent Str alleles. Quantitative analysis of sterols from tissues of affected Bpa mice support a role for Nsdhl in cholesterol biosynthesis. Our results demonstrate that Bpa and Str are allelic mutations and identify the first mammalian locus associated with an X-linked dominant, male-lethal phenotype. They also expand the spectrum of phenotypes associated with abnormalities of cholesterol metabolism.

Four ubiquitously expressed genes, RD (D6S45)-SKI2W (SKIV2L)-DOM3Z-RP1 (D6S60E), are present between complement component genes factor B and C4 in the class III region of the HLA.

The association of the HLA class III region with many diseases motivates the investigation of unidentified genes in the 30-kb segment between complement component genes Bf and C4. RD, which codes for a putative RNA binding protein, is 205 bp downstream of Bf. SKI2W (HGMW-approved symbol SKIV2L), a DEVH-box gene probably involved in RNA turnover, is 171 bp downstream of RD (HGMW-approved symbol D6S45). RP1 (HGMW-approved symbol D6S60E) is located 611 bp upstream of C4. The DNA sequence between human RD and RP1 was determined and the exon-intron structure of SKI2W elucidated. SKI2W consists of 28 exons. The putative RNA helicase domain of Ski2w is encoded by 9 exons. Further analysis of the 2.5-kb intergenic sequence between SKI2W and RP1 led to the discovery of DOM3Z. The full-length cDNA sequence of DOM3Z encodes 396 amino acids with a leucine zipper motif. Dom3z-related proteins are present in simple and complex eukaryotes. In Caenorhabditis elegans, Dom3z-related protein could be involved in the development of germ cells. Human RD-SKI2W and DOM3Z-RP1 are arranged as two head-to-head oriented gene pairs with unmethylated CpG sequences at the common 5' regulatory region of each gene pair. The ubiquitous expression pattern suggests that these four genes are probably housekeeping genes.

The human DEVH-box protein Ski2w from the HLA is localized in nucleoli and ribosomes.

The human helicase gene SKI2W is located between RD and RP1 in the class III region of the major histocompatibility complex. Transcripts of SKI2W are detectable in RNA samples isolated from multiple tissues. The protein product Ski2w shares striking amino acid sequence similarities to the yeast antiviral protein Ski2p that controls the translation of mRNAs, probably based on the mRNA structural integrity. Whether this translational regulation mechanism for cellular and viral RNAs exists in mammals is under investigation. Antisera against human Ski2w were generated using fusion proteins produced in bacteria or insect cells. Western blot analysis showed that the endogenous Ski2w protein is approximately 140 kDa in size and is enriched in polysomal fractions of cytoplasmic extracts from HeLa cells. Ribosomal profile studies revealed that Ski2w distributed throughout the entire sucrose gradient in the presence of Mg2+, but co-sedimented with the 18S rRNA-containing 40S subunit and the small ribosomal subunit protein S27a in the presence of EDTA. The co-sedimentation of Ski2w with the 40S subunit is not affected by RNase A treatment of the cell extract, or the addition of KCl to 0.5 M, suggesting that Ski2w is associated with the 40S ribosomal subunit. Indirect immunofluorescence experiments showed that human Ski2w is localized in the nucleoli and in the cytoplasm. In essence, human Ski2w is present at the sites of ribosome biogenesis and protein synthesis.

Human helicase gene SKI2W in the HLA class III region exhibits striking structural similarities to the yeast antiviral gene SKI2 and to the human gene KIAA0052: emergence of a new gene family.

Helicases are essential enzymes for life because DNA replication, DNA repair, recombination, transcription, RNA splicing and translation all involve more than one helicase to unwind DNA or RNA. We have discovered, cloned and partially characterized a novel human helicase gene, SKI2W. The human SKI2W is located between the RD and RP1 genes in the class III region of the major histocompatibility complex (MHC) on chromosome 6, a genomic region associated with many malignant, genetic and autoimmune diseases. Derived amino acid sequence of human SKI2W showed an open reading frame for 1246 residues. It contains consensus sequences for structural motifs of an RNA helicase with a DEVH box. It has a leucine zipper motif that may be important for protein dimerization, and an RGD motif close to the N-terminus that might serve as a ligand for integrin or cell adhesion molecules. SKI2W shares a striking and extensive similarity to the yeast Ski2p that is involved in the inhibition of translation of poly(A) negative [poly(A)-] RNA, and plays an important role in antiviral activities. Human SKI2W fusion protein expressed in insect cells using a baculovirus vector has ATPase activity. The human SKI2W protein and the yeast Ski2p share extensive sequence similarities to another putative human protein KIAA0052, suggesting the presence of a new gene family that may be involved in translational regulation of cellular and viral RNA.

Complement component C4 gene intron 9 as a phylogenetic marker for primates: long terminal repeats of the endogenous retrovirus ERV-K(C4) are a molecular clock of evolution.

The complement component C4 genes of Old World primates exhibit a long/short dichotomous size variation, except that chimpanzee and gorilla only contain short C4 genes. In human it has been shown that the long C4 gene is attributed to the integration of an endogenous retrovirus, HERV-K(C4), into intron 9. This 6.36 kilobase retroviral element is absent in short C4 genes. Here it is shown that the homologous endogenous retrovirus, ERV-K(C4), is present precisely at the same position in the long C4 gene of orangutan and African green monkey. Determination of the short C4 gene intron 9 sequences from human, three apes, two Old World monkeys, and a New World monkey allowed the establishment of consistent phylogenetic trees for primates, which favors a chimpanzee-gorilla clade. The 5' long terminal repeats (LTR) and 3' LTR of ERV-K(C4) in long C4 genes of human, orangutan, and African green monkey have similar sequence divergence values of 9.1%-10.5%. These values are more than five-fold higher than the sequence divergence of the homologous intron 9 sequences between the long and short C4 genes in higher primates. The latter is probably a result of homogenization or concerted evolution. We suggest that the 5' LTR and 3' LTR of an endogenous retrovirus can serve as a reliable reference point or a molecular clock for studies of gene duplication and gene evolution. This is because the 5'/3' LTR sequences were identical at the time of retroviral integration and evolved independently of each other afterwards. Our data provides strong evidence for the short C4 gene being the ancestral form in primates, trans-species evolution, and the "slow-down" phenomenon of the sequence divergence in great apes.

Structure and genetics of the partially duplicated gene RP located immediately upstream of the complement C4A and the C4B genes in the HLA class III region. Molecular cloning, exon-intron structure, composite retroposon, and breakpoint of gene duplication.

The correlation of many HLA-associated autoimmune and genetic diseases with the polymorphic complement C4 genes may be attributed to the presence of disease susceptibility genes in the close proximity of C4. We have cloned and characterized a pair of partially duplicated genes, RP1 and RP2, located 611 base pairs upstream of the human C4A and C4B genes, respectively. The putative RP protein, consisting of 364 amino acid residues, is basic and highly hydrophilic. There is a bipartite nuclear localization signal at residues 114-131 and therefore RP may be a nuclear protein. Northern blot analysis suggested that RP is ubiquitously expressed. The 5' region of the RP1 gene is CpG rich, which is a characteristic of housekeeping genes. The RP1 gene contains nine exons. Located in the fourth intron is a cluster of Alu elements, and a newly defined composite retroposon SVA with a SINE, multiple copies of GC-rich VNTRs and an Alu element altogether enclosed by direct terminal repeats. Members of SVA are also present in the complement C2 gene located about 20 kilobases upstream of RP1 in the HLA and in the cytochrome CYP1A1 gene. Determination of the DNA sequences for RP2 from two different HLA haplotypes revealed identical hybrid sequences which resulted from fusion of RP with the tenascin-like Gene X and truncation of the 5' regions of both genes. Cumulative data suggest that the four tandemly arranged genes RP, complement C4, steroid 21-hydroxylase (CYP21), and Gene X altogether form a modular structure, RCCX. The number of RCCX modules varies from one to three or more in the population. Absence of the truncated genes RP2 and Gene XA have been detected in genomes with single RCCX modules. Duplication of the RCCX modules probably occurred before the speciation of great apes and humans as they contain the same breakpoint region of RP and Gene X gene duplication.

The dichotomous size variation of human complement C4 genes is mediated by a novel family of endogenous retroviruses, which also establishes species-specific genomic patterns among Old World primates.

The human complement C4 genes in the HLA exhibit an unusual, dichotomous size polymorphism and a four-gene, modular variation involving novel gene RP, complement C4, steroid 21-hydroxylase (CYP21), and tenascin-like Gene X (RCCX). The C4 gene size dichotomy is mediated by an endogenous retrovirus, HERV-K(C4). Nearly identical sequences for this retrotransposon are present precisely at the same location in the long C4 genes from the tandem RCCX Module I and Module II. Specific nucleotide substitutions between the long and short C4 genes have been identified and used for diagnosis. Southern blot analyses revealed that HERV-K(C4) is present at more than 30 locations in the human genome, exhibits variations in the population, and its analogs exist in the genomes of Old World primates with species-specific patterns. Evidence of intrachromosomal recombination between the two long terminal repeats of HERV-K(C4) is found near the huntingtin locus on chromosome 4. It is possible that members of HERV-K(C4) are involved in genetic instabilities including the RCCX modules, and in protecting the host genome from retroviral attack through an antisense strategy.

The transforming prototype of Epstein-Barr virus (B95-8) is also a lytic virus.

The B95-8 isolate of the Epstein-Barr virus (EBV) has been described as a non-lytic transforming virus. We have performed experiments in order to determine if the B95-8 EBV is capable of super-infecting and replicating in EBV-genome-positive non-producer lymphoblastoid cells. Using concentrates of B95-8 EBV, prepared from 6 different B95-8 cell lines treated with 12-O-tetradecanoylphorbol-13-acetate (TPA), we demonstrated that virus concentrates could transform human or cotton-top tamarin B-lymphocytes and also lytically replicate in Raji cells, inducing EBV antigens and infectious virus. While the virus obtained from B95-8 super-infected Raji cells was able to transform cord-blood lymphocytes (CBLs) and super-infect Raji cells, transformation was abortive, with cell cultures only growing for up to 6 weeks. Transformation titers of the B95-8 virus concentrates ranged from 10(5) to greater than 10(8) transforming units/ml; early antigen (EA) induction ranged from 1% to 50% after superinfection of Raji cells, depending on the virus stock used, as determined by immunofluorescence. Southern blot analysis was carried out on the DNA prepared from B95-8 cells and virion DNA. The results were consistent with the published EcoRI restriction pattern for B95-8 EBV. The issue of whether the B95-8 cells produce virions with a dual biological phenotype or, rather, 2 biologically distinct viruses, is addressed.