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A strategy is described that allows the development of polymorphic genetic markers to be characterized in individual genes. Segments of the 3' untranslated regions are amplified, and polymorphisms are detected by digestion with frequently cutting enzymes and with the detection of single-stranded conformation polymorphisms. This allows these genes, or DNA segments, to be placed on the linkage maps of human chromosomes. Polymorphisms in two genes have been identified using this approach. A HaeIII polymorphism was detected in the KIT proto-oncogene, physically assigned to chromosome 4q11-12. This polymorphism is linked to other chromosome 4p markers and is in linkage disequilibrium with a HindIII polymorphism previously described at this locus. We have also identified in the insulin-like growth factor1 receptor gene (IGF1R) a 2-bp deletion that is present at a frequency of .25 in the Caucasian population. Pedigree analysis with this insertion/deletion polymorphism placed the IGF1R gene at the end of the current linkage map of chromosome 15q, consistent with the physical assignment of 15q2526. Thus, polymorphisms in specific genes can be used to related the physical, genetic, and comparative maps of mammalian genomes and to simplify the testing of candidate genes for human diseases.

We previously reported the isolation and characterization of a cDNA clone, I-309, that encodes a small secreted protein produced by activated human T lymphocytes. This protein is structurally related to a large number of recently identified proteins that are secreted upon cellular activation. In this report we describe the isolation and characterization of the gene encoding I-309. The genomic organization is essentially identical to that found in the genes encoding the structurally similar proteins TCA-3, hJE/MCP-1, and mJE, strengthening the hypothesis that these genes are evolutionarily related. The region of the I-309 gene 5' of the mRNA cap site exhibits extensive nucleotide sequence homology with the same region of the murine gene TCA-3, providing additional evidence that I-309 and TCA-3 are likely to be homologs. Finally, panels of rodent-human somatic cell hybrids were used to map the I-309 gene to human chromosome 17. In conjunction with recent mapping data from other laboratories, this result suggests the presence of a cluster of related genes on this chromosome.

We have assigned the human serum amyloid A (SAA) gene family to a 90 kb region on the short arm of human chromosome 11 (11p) by hybridization of defined genomic fragments of human SAA genes to DNA from rodent-human somatic cell hybrids and to large DNA fragments separated by transverse alternating field gel electrophoresis. We have also characterized SAA probe hybridization patterns in human DNA cleaved with restriction endonucleases Hind III, Pst I, BglII, TaqI, and XbaI and found invariant patterns except for a two-allele restriction fragment length polymorphism (RFLP) with Hind III. These studies show that the SAA gene family comprises at least three members in the haploid human genome and will be useful in identifying variant patterns and establishing linkage between members of the SAA gene family and other markers on chromosome 11.

T-cell growth factor (TCGF) or interleukin-2 (IL-2), an immunoregulatory lymphokine, is produced by lectin- or antigen-activated mature T lymphocytes and in a constitutive manner by certain T-cell lymphoma cell lines. By means of a molecular clone of human TCGF and DNA extracted from a panel of somatic cell hybrids (rodent cells X normal human lymphocytes), the TCGF structural gene was identified on human chromosome 4. In situ hybridization of the TCGF clone to human chromosomes resulted in significant labeling of the midportion of the long arm of chromosome 4, indicating that the TCGF gene was located at band q26-28. Genomic DNA from a panel of hybrids prepared with HUT-102 B2 cells was examined with the same molecular clone. In this clone of cells, which produces human T-cell leukemia virus, the TCGF gene was also located on chromosome 4 and was apparently not rearranged. The homologous TCGF locus in the domestic cat was assigned to chromosome B1 by using a somatic cell hybrid panel that segregates cat chromosomes. Linkage studies as well as high-resolution G-trypsin banding indicate that this feline chromosome is partially homologous to human chromosome 4.

The human dihydrofolate reductase (DHFR; tetrahydrofolate dehydrogenase; 5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3) gene family includes a functional gene (hDHFR) and at least four intronless genes. Three intronless genes (hDHFR-psi 2, hDHFR-psi 3, and hDHFR-psi 4) are identifiable as pseudogenes because of DNA sequence divergence from the functional gene with introns, while one intronless gene (hDHFR-psi 1) is completely homologous to the coding sequences of the functional gene. Analysis of genomic DNA from two panels of somatic human-rodent cell hybrids with specific molecular probes provide insight into the chromosomal organization and assignment of these genes. The five genes are dispersed in that each one is found on a different chromosome. The functional gene hDHFR has been assigned to chromosome 5, and one pseudogene (hDHFR-psi 4), to chromosome 3. In a human cell line (HeLa) that was selected for methotrexate resistance, the functional locus became amplified, while there was no amplification of the four intronless pseudogenes. hDHFR-psi 1 was found to be present in DNA of some individuals and absent from DNA of others, consistent with a recent evolutionary origin of this gene originally suggested by its sequence identity to the coding portions of the functional gene. The presence or absence of this intronless pseudogene represents a previously unreported form of DNA polymorphism.

The electrophoretic resolution of a group of genetically monomorphic gene-enzyme systems that are developmentally and biologically ubiquitous has been used to provide a species-specific and type-specific biochemical characterization of various cultured cells. The relative mobilities of gene-enzyme systems representing nine distinct gene products from cell cultures of 25 species from Drosophila to man are presented. These isoenzymes effectively discriminate interspecies cell-to-cell contamination and almost invariably serve to identify the contaminating species. The resolution of eight polymorphic gene-enzyme systems in human cell cultures provides a virtually unique allozyme genetic signature as a monitor of intraspecies cellular contamination. The genetic signatures of 47 commonly used human cells are presented. Included in the test were seven putative HeLa (human cervical carcinoma) contaminants each of which expressed a signature identical with that of HeLa. The probability that an unrelated human cell line will have a signature identical to a typed cell is computed for each line from the genotypic frequencies at each locus in a population of cultured human cells. The gene frequencies of this cell population are comparable to the same frequencies in natural human populations. The most common human signature has a frequency (and therefore a probability) of 0.02. The majority of the 17,010 possible signatures are far less probable. A calculation of the theoretical incidence of chance matching of signatures within test groups of two or more individuals is presented. The probability of a chance match between any two randomly selected individuals is 0.004 and among five randomly selected individuals is 0.034. The allozyme genetic signature represents a definitive monitor of cell identity and is presented as a standard of cell and tissue identification for a variety of biological studies.

Siamese cats are homozygous for the recessive cs allele of the color (albino) locus. The c locus is shown here by backcross analysis to be linked to the beta-hemoglobin (HBB) locus in the cat at a distance of approximately eight centiMorgans. The HBB locus and, by inference, the c locus were assigned to feline chromosome D1, by analysis of genomic DNAs from a panel of rodent X cat somatic cell hybrids with a molecular clone of the human beta-globin locus. Evolutionary conservation of the synthetic homology of feline chromosome D1 and human chromosome 11 is extensive. Comparison of high resolution G-trypsin-banded preparations of the two chromosomes permitted cytological alignment of the long arm of the conserved chromosomes providing that a minimum of one paracentric inversion is hypothesized. The placement of the albino locus on conserved syntenic groups of several markers (HBB, HRAS, LDHA) in both cat and mouse strongly indicates the conservative placement of the as yet unmapped human albino locus in the homologous syntenic group on human chromosome 11p.

A panel of 42 rodent x cat somatic cell hybrids has been used to assign seven structural genes for lysosomal enzymes to specific chromosomes in the domestic cat. The assignments include alpha-glucosidase (GANAB) to chromosome D1, alpha-galactosidase (GLA) to the X chromosome, beta-galactosidase 1 (GLB1) to chromosome B3, beta-glucuronidase (GUSB) to chromosome E3, alpha-mannosidase A (MANA) to chromosome B3, alpha-L-fucosidase (FUCA) to chromosome C1, and hexosaminidase A (HEXA) to chromosome B3. In all cases, the feline lysosomal enzyme genes were located in linkage groups which were syntenic with their homologous positions in the human gene map. These assignments expand the genetic map of the cat and reaffirm the extensive syntenic homology between the chromosome maps of man and cat.

Previous characterization of GLI, a gene found to be amplified and expressed in a subset of human brain tumors, revealed the presence of five tandem zinc fingers related to those of Krüppel (Kr), a Drosophila segmentation gene of the gap class. We have used the GLI cDNA as a molecular probe to isolate related sequences from the human genome. Partial characterization of six related loci, including sequence determination, expression studies, and chromosome localization, revealed that each locus could encode a separate finger protein. The predicted proteins all had similar H-C links, i.e., a conserved stretch of 9 amino acids connecting the C-terminal histidine of one finger to the N-terminal cysteine of the next. On the basis of amino acid sequence and intron-exon organization, the genes could be placed into one of two subgroups: the GLI subgroup (with the consensus finger amino acid sequence [Y/F]XCX3GCX3[F/Y]X5LX2HX3-4H[T/S]GEKP) or the Kr subgroup (with the consensus finger amino acid sequence [Y/F]XCX2CX3FX5LX2HXRXHTGEKP). Unlike GLI or Kr, most of the newly isolated genes were expressed in many adult tissues. The predicted proteins probably control the expression of other genes and, by analogy with Kr and GLI, may be important in human development, tissue-specific differentiation, or neoplasia.

We have isolated a 2.1-kb cDNA clone from a human placental library encoding part of the alpha 2 chain of collagen IV, a major structural protein of basement membranes. The DNA sequence encodes 446 amino acids in the triple-helical domain plus the 227 amino acids of the carboxy-terminal globular domain. The latter structure is composed of two homologous subdomains and is highly conserved between the alpha 1 and alpha 2 chains. The triple-helical domain contained seven interruptions of the Gly-X-Y repeat and these interruptions were in general larger than their counterparts in the alpha 1 chain. DNA from human rodent hybrid cell lines was analyzed under conditions in which there was no cross-hybridization of the alpha 2(IV) cDNA probe with the gene for the alpha 1(IV) collagen chain. An EcoRI fragment characteristic of the alpha 2 chain had a concordance of 0.97 with chromosome 13. This result was confirmed and extended with in situ localization of the gene at 13q34. Since the alpha 1(IV) gene has previously been localized to 13q34, the two type IV collagen genes reside in the same chromosome region (13q34), possibly in a gene cluster. The presence of the genes for type IV collagen chains on chromosome 13 excludes a primary role for these genes in adult polycystic kidney disease and X-linked forms of hereditary nephritis.

The human interleukin 1 alpha gene was assigned to chromosome 2 using Southern transfer analysis of human-rodent somatic cell hybrid DNAs. The gene was regionally localized to 2q12-21 using in situ hybridization to metaphase chromosomes. These results indicate that the IL-1 alpha gene maps to the same general region on the long arm of chromosome 2 as the IL-1 beta gene, which has been previously assigned.

Bvr-1 is a dominant X-linked feline gene which restricts the replication of B-tropic murineleukemia virus (B-MuLV) in somatic cell hybrids between murine BALB/c-RAG cells and FL-74 feline cells. Since the hybrids were originally derived by the hypoxanthine aminopterin thymidine selection scheme, counter selection experiments on 6-thioguanine result in preferential survival of hybrid cells which have spontaneously lost the feline X-chromosome on which is located the structural gene for hypoxanthine guanine phosphoribosyl transferase (IMP: pyrophosphate phosphoribosyl transferase, E.C. 2.4.2.8) and Bvr-1. Back selected Bvr-1- cells express high parental levels of B-MuLV. Bvr-1 effectively restricts the IdU-mediated induction of the endogenous xenotropic BALB virus (BALB: virus 2) but not the endogenous N-tropic virus (BALB: virus 1). Pleiotropic restriction of B-MuLV and X-MuLV, but not N-MuLV suggests that the viral targets of Bvr-1 (either viral components or functions in viral assembly) of the B-tropic and X-tropic endogenous BALB viruses are similar to each other but distinct from the target in the N-tropic virus. Very low levels of B-MuLV are detected in restricted cells, but this residual virus is not infectious in either NIH-3T3 or BALB-3T3 mouse cells which are genotypically Fv-1N/Fv-1N and Fv-1B/Fv-1B, respectively. Passage of residual virus through host cells without Fv-1 related restriction (SC-1) results in production of infectious B-MuLV indistinguishable from that produced by RAG parent cells.

A relatively recently identified family of structurally similar Ca2(+)-dependent phospholipid binding proteins is called the annexin gene family. At least seven genes are known, although their exact functions are unclear. The endonexin II gene (ENX2), one member of the gene family, is assigned to 4q28----q32 using both Southern transfer analysis of human x rodent somatic cell hybrid DNAs and in situ chromosome hybridization. One of the lipocortin II genes, another annexin, had previously been assigned to the long arm of chromosome 4.

A method for detecting possible structural genes in D. melanogaster based on gene dosage dependency is presented. By making thirty crosses between Y-autosome translocations, and an attached-4 cross, it is possible to produce large duplications (approximately 150 salivary gland chromosome bands in length) for every autosomal region with the exception of 83DE. The usefulness of the technique was demonstrated by dosage dependency of three known gene-enzyme systems: alpha-glycerophosphate dehydrogenase-1, alcohol dehydrogenase and malate dehydrogenase. A screen for genes affecting two enzymes localized on the inner membrane of the mitochondrion, alpha-glycerophosphate oxidase (alphaGPO) and succinic dehydrogenase (SHD), produced a dosage-sensitive region in each case. Region 50C-52E affected alphaGPO activity and region 28D-29F affected SDH activity. The latter region apparently includes the malic dehydrogenase-1 gene. The methodology and limitations of the technique are discussed.

The stromal-derived factor-1 (SDF-1) chemokine gene encodes the only natural ligand for CXCR4, the coreceptor for the pathogenic X4 HIV-1 strains. A single-nucleotide polymorphism (SNP) in the 3′ untranslated region (SDF1-3′A=rs1801157) of SDF-1 was reported to be protective against infection and progression in some, but not other, epidemiological studies. To identify additional alleles that may influence HIV-1 infection and progression to AIDS, nine SNPs (including rs1801157) spanning 20.2 kb in and around the SDF-1 gene were genotyped in over 3000 African American (AA) and European American (EA) participants enrolled in five longitudinal HIV-1/AIDS natural cohort studies. Six or five haplotypes were present at frequencies greater than 5% in AA or EA, respectively. Six of the nine SNPs occur on only one common haplotype (>5%), while the remaining three SNPs were found on multiple haplotypes, suggesting a complex history of recombination. Among EA, rs754618 was associated with an increased risk of infection (OR=1.50, P=0.03), while rs1801157 (=SDF1-3′A) was associated with protection against infection (OR=0.63, P=0.01). In the MACS cohort, rs1801157 was associated with AIDS-87 (RH=0.31, P=0.02) and with death (RH=0.18, P=0.02). Significant associations to a single disease outcome were found for two SNPs and one haplotype in AA.Genes and Immunity (2005) 6, 691–698. doi:10.1038/sj.gene.6364258; published online 22 September 2005 [ABSTRACT FROM AUTHOR]

The polymorphic MHC class I chain-related A (MICA) gene encodes a ligand that has different binding affinities for the NKG2D activating receptor of CD8+ T cells and natural killer (NK) cells. We hypothesized that MICA heterogeneity would affect recovery from hepatitis C virus (HCV) and hepatitis B virus (HBV) infections. To test the hypothesis, we initially typed known MICA polymorphisms for 228 persons who cleared HCV infection and 442 persons with persistent hepatitis C matched on other factors affecting viral persistence. Although MICA*015 was detected more than two-fold more often in persons with viral clearance (odds ratio 0.36, 95% confidence interval=0.19, 0.80), it occurred in fewer than 5% of the study population. In a similar analysis of 442 persons with chronic hepatitis B and 768 matched controls who recovered, MICA*015 was detected in 2.0% of persons with chronic hepatitis B and only 0.9% of controls. No significant associations were detected with other MICA polymorphisms. While further investigation may reveal a structural basis of the MICA*015 associations, these data provide little support for the hypothesis that differential distribution of MICA alleles substantially affects recovery from HCV and HBV infections.Genes and Immunity (2004) 5, 261-266. doi:10.1038/sj.gene.6364065 Published online 18 March 2004 [ABSTRACT FROM AUTHOR]

Previous research has demonstrated isolated effects of host genetic factors on the progression of human immunodeficiency virus type 1 (HIV-1) infection. In this paper, the authors present a novel use of multivariable methods for estimating the prevented fraction of acquired immunodeficiency syndrome (AIDS) cases attributable to six restriction genes after accounting for their epidemiologic interactions. The methods presented will never yield a prevented fraction above 1. The study population consisted of a well-characterized cohort of 525 US men with HIV-1 seroconversion documented during follow-up (1984–1996). On the basis of a regression tree approach using a Cox proportional hazards model for times to clinical AIDS, the combinations of genes associated with the greatest protection, relative to the lack of a protective genotype, consisted of: 1) C-C chemokine receptor 5 (CCR5)-Δ32and C-C chemokine receptor 2 (CCR2)-64I (relative hazard = 0.44); 2) interleukin 10 (IL10)-+/+ in combination with CCR5-Δ32orCCR2-64I (relative hazard = 0.45); and 3) IL10-+/+ in combination with stromal-derived factor (SDF1)-3 ′A and CCR5 promoter P1/~P1 (relative hazard = 0.37). Overall, 30% of potential AIDS cases were prevented by the observed combinations of restriction genes (95% confidence interval: 7, 47). However, the combined effect was confined to the first 4 years following HIV-1 seroconversion. Additional research is needed to identify AIDS restriction genes with stronger and long-lasting protection to better characterize the genetic epidemiology of HIV-1. [ABSTRACT FROM AUTHOR]

Since the discovery of Viliuisk encephalomyelitis (VE) in 1887, scientists have tried to understand the natural history and aetiology of this endemic neurological disorder among the native Sakha population of Central Siberia. Familial aggregation and segregation analysis suggested a genetic influence on VE incidence. However, recent studies have implicated an unknown virus, possibly from the alpha herpesvirus family, as a possible cause for this disease. As VE is a neurological disease characterized by the inflammatory reactions systematically observed in the spinocerebellar fluid and in the brain tissue of deceased patients, we examined 17 single nucleotide polymorphisms (SNPs) across seven inflammation-related candidate gene regions, including chemokine receptors type 2 and 5 ( CCR2/CCR5), interferon-γ ( IFN-γ), interleukin-4 ( IL-4), IL-6, IL-10, stromal cell-derived factor ( SDF) and chemokine regulated upon activation, normal T-cell expressed and presumably secreted ( RANTES). Our main objective was to analyse the degree of genetic association between VE and candidate genes that have been previously implicated in other inflammatory diseases. Samples were collected from 83 affected families comprising 88 verified VE cases, 156 family members, and an additional 69 unrelated, unaffected inhabitants of the same geographical area. This collection included substantially all of the cases that are currently on the VE Registry. The experimental design included both case–control and transmission/disequilibrium test (TDT)-based familial association analyses. None of 17 SNPs analysed was significantly associated with VE occurrence. Exclusion of these eight genes based on the lack of association has important implications for identifying the disease agent, as well as prescribing therapy and understanding Viliuisk encephalomyelitis. [ABSTRACT FROM AUTHOR]