Sequence analysis and mapping of the Sry gene in species of the subfamily Arvicolinae (rodentia).

The rodent subfamily Arvicolinae, which contains about 125 species, presents some interesting exceptions concerning Sry, the sex determining gene in mammals. In some species multiple Sry copies have been described on the Y chromosome and in the Iberian vole, Microtus cabrerae, several Sry sequences have been cloned and mapped not only on the Y but also on the X chromosome. Here we present a comparative analysis of Sry sequences from a total of 22 species. Our study demonstrates for the first time that for most North American species, as previously reported for the European species, multiple copies of the Sry gene exist on the Y chromosome. Furthermore, we have sequenced and analyzed the full sequence of Sry from several European species, showing that the sequence and structure of the gene in this group of species present the main features described for Sry in other mammals. Finally, FISH analyses on some of these species demonstrated that all Sry sequences, despite their functional status, mapped on the euchromatic short arm of the Y chromosome.

Molecular cytogenetics of the california condor: evolutionary and conservation implications.

Evolutionary cytogenetic comparisons involved 5 species of birds (California condor, chicken, zebra finch, collared flycatcher and black stork) belonging to divergent taxonomic orders. Seventy-four clones from a condor BAC library containing 80 genes were mapped to condor chromosomes using FISH, and 15 clones containing 16 genes were mapped to the stork Z chromosome. Maps for chicken and finch were derived from genome sequence databases, and that for flycatcher from the published literature. Gene content and gene order were highly conserved when individual condor, chicken, and zebra finch autosomes were compared, confirming that these species largely retain karyotypes close to the ancestral condition for neognathous birds. However, several differences were noted: zebra finch chromosomes 1 and 1A are homologous to condor and chicken chromosomes 1, the CHUNK1 gene appears to have transposed on condor chromosome 1, condor chromosomes 4 and 9 and zebra finch chromosomes 4 and 4A are homologous to chicken chromosome arms 4q and 4p, and novel inversions on chromosomes 4, 12 and 13 were found. Condor and stork Z chromosome gene orders are collinear and differentiated by a series of inversions/transpositions when compared to chicken, zebra finch, or flycatcher; phylogenetic analyses suggest independent rearrangement along the chicken, finch, and flycatcher lineages.

Haplotype analysis of the SDF-1 (CXCL12) gene in a longitudinal HIV-1/AIDS cohort study.

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.

Morphological, chromosomal, and molecular evolution are uncoupled in pocket mice.

C-, and G-banded chromosomes are presented for Perognathus amplus and Perognathus longimembris from Arizona, USA and Chaetodipus nelsoni from Coahuila, Mexico. The two species of Perognathus reveal similar C-band patterns, and extensive autosomal and X chromosome G-band identity with only pericentric inversions distinguishing pairs 4 and 6 and a difference in the morphology of pair 20. Three pairs of autosomal secondary constrictions were found in P. amplus and only one in P. longimembris. Only 50% of the amplus/longimembris G-banded karyotype could be aligned with that of C. nelsoni indicating extensive chromosomal restructuring has taken place since these genera last shared a common ancestor. A review of the literature suggests variable rates of morphological, chromosomal and molecular evolution in these animals.

The human MIP-1beta chemokine is encoded by two paralogous genes, ACT-2 and LAG-1.

Human macrophage inflammatory protein-1 beta (MIP-1beta) is an Mr 8,000 acidic protein that is upregulated upon stimulation in monocytes, T cells, and other lymphocytes. This protein belongs to the CC chemokine subfamily and directs the migration of specific subsets of leukocytes. The first molecular clone was isolated in 1988, and ever since there has been confusion regarding the exact number of genes encoding this and closely related proteins. PCR primers were designed from two genomic GenBank entries to conduct single-strand conformational polymorphism analysis, sequence analysis, and PCR-RFLP, and we conclude that previously isolated clones referred to as MIP-1beta are derived from two genes, originally called ACT-2 and LAG-1. The two proteins share a common length and are identical at 89 of 92 amino acids. The first two amino acid differences, V12M and L20P, occur in the signal peptide, while the third, G70S, is in the mature protein. Within the transcribed region, the genes differ at 25 of 662 nucleotides. A survey of the NCBI expressed sequence tag database reveals that both genes are expressed in a variety of tissues, and five clones representing LAG-1 transcripts are alternatively spliced, with the 115-bp exon 2 omitted. Database searches for putative orthologues in other species revealed that the rabbit protein is about 80% similar to the two human proteins, while those of rat and mouse are 70-75% similar. Comparative sequence analysis of the human and animal proteins indicates substantially higher rates of protein evolution in the two rodents compared to human and rabbit.

Stable methylation patterns in interspecific antelope hybrids and the characterization and localization of a satellite fraction in the Alcelaphini and Hippotragini.

Conflicting data has recently appeared concerning altered methylation patterns in interspecific mammalian hybrids and the potential this may hold for driving karyotypic evolution. We report no detectable methylation difference in the genomic DNA of different interspecific F1 antelope hybrids (family Bovidae) and their parent species using the methylation-sensitive enzyme HpaII and its methylation insensitive isoschizomer MspI. However, both enzymes released a tandemly repeated satellite array. Characterization of the repeat using Southern blotting and a combination of sequencing, fluorescence in-situ hybridization (FISH) and C-banding, shows some similarity in the family of repeats between the hybridizing antelope species groups, and that the satellite is localized in the centromeric C-band positive regions of the chromosomes. Moreover, although there is little meaningful sequence homology with the well characterized bovine 1.715 satellite DNA, there is 86% sequence similarity with the sheep/goat satellite I, suggesting that they are related and are likely to have originated and evolved separately from the bovine unit.

DRM/GREMLIN (CKTSF1B1) maps to human chromosome 15 and is highly expressed in adult and fetal brain.

We have mapped and characterized the human homolog of Drm/Gremlin (CKTFS1B1), a member of a family of BMP antagonists that have been linked to both developmental and transformation-related functions. By screening a human cDNA library, we isolated a 3.3-kb cDNA containing the 552-bp region encoding the human DRM protein. CKTFS1B1 was localized on human chromosome 15q13--> q15 by somatic cell hybrid analysis and, more precisely, using radiation hybrids, to a region of markers linked to SGNE1, secretory granule neuroendocrine protein 1 and RYR3, the ryanodyne receptor 3. Northern blot analysis showed the presence of a single DRM-specific mRNA expressed in different human tissues, including brain, ovary, intestine and colon. In the brain, DRM expression is associated with the region localized around the internal capsule in the large subcortical nuclei. DRM appears to be predominantly expressed in normal cells and tissues, including normal neurons, astrocytes and fibroblasts. Interestingly, we detected DRM expression in normal cells obtained from several patients, but not in tumor cell lines established from the same patients. The data suggest that down-regulation of DRM is associated with tumor progression, and support the hypothesis that human DRM may play an important role during both neuroembryological development and carcinogenesis.

Polymorphisms of the human IFNG gene noncoding regions.

Interferon gamma (IFN-gamma) is a multifunctional cytokine that is essential in the development of Th1 cells and in cellular responses to a variety of intracellular pathogens including human immunodeficiency virus (HIV-1). We screened genomic DNA samples from a predominately Caucasian male population of HIV-infected and healthy donors for polymorphisms in the human IFNG gene from -777 to +5608 by single-stranded conformational polymorphism. Surprisingly, the proximal promoter (-777 to transcription start) is invariant as no polymorphisms were found in over 100 samples tested. However, further screening revealed polymorphisms in other regions of the gene including a single base insertion in a poly-T tract in the first intron, three single base pair substitutions in the third intron, and another single base pair substitution in the 3' untranslated region (UTR). Electrophoretic mobility shift assay was used to investigate whether these variants have altered DNA-binding abilities, since intronic enhancer elements have been reported for the IFNG gene. Oligonucleotides constructed for two third intron variants showed no difference in DNA-binding abilities as compared with wild-type sequences. However, the 3'UTR variant showed the formation of unique DNA-binding complexes to radiolabeled oligonucleotide probes as compared with the wild-type sequence. The influence of a CA-repeat microsatellite on AIDS disease progression in HIV-1 seroconverters was tested by a Cox proportional hazards model. There is no evidence of an association between alleles and infection with HIV-1 or progression to AIDS. We report an invariant proximal human IFNG promoter and the existence of multiple intronic variants and a potentially functional 3'UTR polymorphism.

Molecular cloning of the guinea pig GRO gene and its rapid expression in the tissues of lipopolysaccharide-injected guinea pigs.

BACKGROUND: CXC chemokines, IL-8 and GRO, play a role in the recruitment of neutrophils in the human. The functional orthologues in the rat and mouse are CINC/KC and MIP-2. The lack of IL-8 made these animals less useful to study the role of IL-8 and GRO. METHODS: Guinea pig (gp) cDNA libraries were screened for GRO and IL-1beta. A gp genomic library was screened with a gpGRO cDNA probe. Expression of gpIL-8, gpGRO, gpTNFalpha, and gpIL-1beta was investigated by Northern analysis and/or by in situ hybridization. RESULTS: Two gpGRO cDNAs, a 3.0-kb gpGRO genomic DNA, and a gpIL-1beta cDNA were cloned. gpGRO and gpIL-8 mRNA were detected in different tissues including lungs 1 h after intraperitoneal injection of lipopolysaccharide (LPS) into guinea pigs. gpGRO, gpIL-8, gpTNFalpha, and gpIL-1beta expression peaked at 3 h in the lungs. Both gpGRO and gpIL-8 mRNA were detected in the cells in alveolar spaces and bronchial epithelial cells. However, gpGRO mRNA, but not gpIL-8, was also expressed in endothelial cells and vascular smooth muscle cells. CONCLUSIONS: gpGRO and gpIL-8 mRNA rapidly accumulated in the lungs of guinea pigs after LPS injection. Expression of gpIL-8 and gpGRO mRNA appeared to be independent from TNFalpha- or IL-1beta-stimulation in this model. A high level expression of gpGRO in vascular cells suggest an important role of GRO in the sequestration of neutrophils and multi-organ injuries induced by LPS. The guinea pig will provide an excellent model to study the roles of IL-8 and GRO, important inflammatory mediators in the human.

Isolation of novel GRO genes and a phylogenetic analysis of the CXC chemokine subfamily in mammals.

Approximately 15 different alpha, or CXC, chemokines have thus far been isolated from 11 species of mammals. Among the best studied chemokines are the 12 human proteins that are encoded by 11 paralogous genes. In order to better understand the evolution and function of this group of genes, we isolated and characterized six novel GRO and GRO-related cDNA sequences from the cow (Bos taurus), the sheep (Ovis aries), the rabbit (Oryctolagus cuniculus), and the guinea pig (Cavia porcellus). The amino acid sequence of the diverged guinea pig GRO or KC gene is only 50%-60% similar to presumed orthologs from other species, while the sheep and cow GRO proteins are 90%-99% similar to each other. The presence of multiple GRO genes in the cow, the rabbit, and the sheep is consistent with what has been observed for humans. Phylogenetic analyses of amino acid sequences from 44 proteins indicate that genes orthologous to many of the 11 known from humans exist in other species. One such gene, interleukin 8, or IL8, has been isolated from nine species, including the rodent guinea pig; however, this gene is absent in the rat and the mouse, indicating a unique gene loss event in the rat/mouse (muroid rodent) lineage. The KC (or MIP2) gene of rodents appears to be orthologous to the GRO gene found in other taxonomic orders. Combined evidence from different sources suggests that IP10 and MIG share sister taxon relationships on the evolutionary tree, while the remaining paralogous genes represent independent lineages, with limited evidence for kinship between them. This observation indicates that these genes originated nearly contemporaneously via a series of gene duplication events. Relative-rate tests for synonymous and nonsynonymous nucleotide substitutions in the KC and IL8 genes did not detect rate heterogeneity; however, there are several notable features regarding the IL8 genes. For example, the IL8 proteins from two Old World monkeys are as similar to one another as they are to the IL8 protein from humans, and all observed nucleotide differences between the IL8 genes of the two monkeys cause amino acid changes; in other words, there are no synonymous differences between them.

Assignment of the mouse and cow CXC chemokine genes.

Gene specific PCR primers were constructed for five mouse and three bovine CXC chemokine genes. The mouse genes were assigned using SSCP analyses of the Jackson BSS backcross panel to two groups on chromosome 5. One group containing Gro1 and Mip2 cosegregated with reference markers Alb1 and Btc, and was positioned 2.2 cM proximal to a group comprising Ifi10, Mig, and Scyb5. The bovine genes IL8, GRO1, and GRO3, mapped using bovine x hamster somatic cell hybrids, were all found to be located on chromosome 6. The locations of these genes in these two animal species are consistent with the positions in humans (4q13-->q21), and previous syntenic relationships among these three mammals.

Isolation and characterization of a bovine neural specific protein (CRMP-2) cDNA homologous to unc-33, a C. elegans gene implicated in axonal outgrowth and guidance.

We have cloned the cDNA encoding bovine CRMP-2 from bovine brains. A full length cDNA encoding bovine CRMP-2 was isolated and sequenced. The deduced amino acid sequence reveals that the gene encodes a protein of 572 amino acids and is highly homologous to Caenorhabditis elegans unc-33, which controls the guidance and outgrowth of neuronal axons. The CRMP-2 transcript was present in bovine brains but not non-neural tissues, and its protein product existed in both soluble and membrane-bound forms. The expression of CRMP-2 protein and mRNA was upregulated during neuronal differentiation of rat PC12 cells. Immunoprecipitation of PC12 cell extracts shows that CRMP-2 was co-immunoprecipitated with a 190 kDa protein (p190). Both CRMP-2 and p190 were phosphorylated on serine residues in vivo and in vitro in a kinase assay of CRMP-2 immune complexes. Immunocytochemistry shows that CRMP-2 was exclusively localized in both the central and peripheral nervous systems in mouse embryos and detectable in the adult brain although the level of CRMP-2 decreased. The protein was expressed in the axon, dendrite, and cytoplasm of postmitotic neurons and in the cytoplasm of oligodendrocytes and astrocytes. The CRMP-2 gene maps to the region of mouse chromosome 14 syntenic with human chromosome 8p21. Taken together, these data suggest multi-functional roles for CRMP-2 in developing and adult nervous systems, and the biological activity of CRMP-2 could be regulated by phosphorylation reaction.

Localization of the human CXC chemokine subfamily on the long arm of chromosome 4 using radiation hybrids.

All 12 of the human CXC chemokine genes were physically mapped using gene-specific PCR primers and the GenBridge 4 radiation hybrid panel. Nine genes, PF4, PF4V1, GRO1, GCP2, PPBP, IL8, GRO2, GRO3, and SCYB5, were assigned within a 1.8-cR interval of one another on 4q. Two additional genes, MIG and INP10, map within 0.5 cR of each another and 6 cR distal to the above-mentioned group. The final gene, SDF1, is localized on 10q. Phylogenetic analyses of amino acid sequences revealed that SDF1 is the most divergent member and that the physically separated MIG-INP10 pair constitutes a distinct evolutionary lineage.

Genetic restriction of AIDS pathogenesis by an SDF-1 chemokine gene variant. ALIVE Study, Hemophilia Growth and Development Study (HGDS), Multicenter AIDS Cohort Study (MACS), Multicenter Hemophilia Cohort Study (MHCS), San Francisco City Cohort (SFCC)

Stromal-derived factor (SDF-1) is the principal ligand for CXCR4, a coreceptor with CD4 for T lymphocyte cell line-tropic human immunodeficiency virus-type 1 (HIV-1). A common polymorphism, SDF1-3'A, was identified in an evolutionarily conserved segment of the 3' untranslated region of the SDF-1 structural gene transcript. In the homozygous state, SDF1-3'A/3'A delays the onset of acquired immunodeficiency syndrome (AIDS), according to a genetic association analysis of 2857 patients enrolled in five AIDS cohort studies. The recessive protective effect of SDF1-3'A was increasingly pronounced in individuals infected with HIV-1 for longer periods, was twice as strong as the dominant genetic restriction of AIDS conferred by CCR5 and CCR2 chemokine receptor variants in these populations, and was complementary with these mutations in delaying the onset of AIDS.

Identification of CCR8: a human monocyte and thymus receptor for the CC chemokine I-309.

The human CC chemokine I-309 is a potent monocyte chemoattractant and inhibits apoptosis in thymic cell lines. Here, we identify a specific human I-309 receptor, and name it CCR8 according to an accepted nomenclature system. The receptor has seven predicted transmembrane domains, is expressed constitutively in monocytes and thymus, and is encoded by a previously reported gene of previously unknown function named, alternatively, CY6, TER1, and CKR-L1. After transfection with the CY6 open reading frame, a mouse pre-B cell line exhibited calcium flux and chemotaxis in response to I-309 (EC50 = 2 nM for each), whereas 20 other chemokines were inactive. Signaling was sensitive to pertussis toxin, suggesting coupling to a Gi-type G protein. These properties parallel those of endogenous I-309 receptors expressed in an HL-60 clone 15 cell line model. The apparent monogamous relationship between I-309 and CCR8 is unusual among known CC chemokines and known CC chemokine receptors. CCR8 may regulate monocyte chemotaxis and thymic cell line apoptosis.

ERF: genomic organization, chromosomal localization and promoter analysis of the human and mouse genes.

ERF (Ets2 Repressor Factor) is a ubiquitously expressed ets-domain protein that exhibits strong transcriptional repressor activity, has been shown to suppress ets-induced transformation and has been suggested to be regulated by MAPK phosphorylation. We report here the sequence of the mouse gene, the genomic organization of the human and the mouse genes, their chromosomal position and the analysis of the promoter region. Genomic clones encompassing either the human ERF or the mouse Erf gene were isolated and utilized to define their molecular organization. The gene in both species consists of 4 exons over a 10 kb region. Utilizing FISH, somatic cell hybrids and linkage analysis, we identified the chromosomal position of ERF on human chromosome 19q13.1 and on its syntenic region in the mouse, on chromosome 7. Sequence analysis of the mouse gene indicated a 90% identity to the human gene within the coding and promoter regions. The predicted Erf protein is 98% identical to the human protein and all of the identifiable motifs are conserved between the two proteins. However, the mouse protein is three amino acids longer (551 versus 548 aa). The area surrounding the region that is homologous to the 5' end of the human cDNA can serve as a promoter in transfection into eukaryotic cells. This region is highly conserved between the mouse and the human genes. A number of conserved transcription factor binding sites can be identified in the region including an ets binding site (EBS). Interestingly, removal of a small segment that includes the EBS, seriously hampers promoter function, suggesting the ERF transcription may be regulated by ets-domain proteins.

The Csk-like proteins Lsk, Hyl, and Matk represent the same Csk homologous kinase (Chk) and are regulated by stem cell factor in the megakaryoblastic cell line MO7e.

Recently, the cDNAs for Lsk, Matk and Hyl, three Csk-related protein tyrosine kinases, have been cloned. We have examined the relationship of Lsk, Matk and Hyl, and found that the gene for each of these proteins is localized to the same region of human chromosome 19. Further, the proteins encoded by Lsk and Matk cDNAs are immunologically similar. These data strongly suggest that Lsk, Hyl and Matk are the same gene product. Previous reports demonstrating expression of Hyl and Matk in hematopoietic lineages led us to investigate the regulation of Lsk expression in response to stem cell factor (SCF) and granulocyte-macrophage colony stimulating factor (GM-CSF) in M07e, a human leukemic cell line. Induction of Lsk/Hyl/Matk protein and mRNA was observed after treatment with SCF but not with GM-CSF. GM-CSF and IL-3, potent mitogens, had no effect on Lsk/Hyl/Matk expression. In contrast, PMA induced Lsk/Hyl/Matk but did not stimulate proliferation. Therefore, induction of Lsk/ Hyl/Matk does not correlate with the capacity to stimulate proliferation. None of the stimuli examined increased Csk protein or mRNA expression. These data demonstrate differential regulation of Csk family members by cytokines and suggest a role for Lsk/ Hyl/Matk in responses mediated by SCF and PMA. Further, our data demonstrate that, as has been seen in blood monocytes, cytokine driven translational control of Lsk/Hyl/ Matk is likely a critical mode of regulation. Lastly, since our studies strongly suggest that the Lsk, Hyl and Matk kinases are related and regulated distinctly from Csk, we and several of the original authors have agreed to rename this kinase the Csk homologous kinase (Chk).

cDNA cloning and characterization of the human interleukin 13 receptor alpha chain.

We have cloned cDNAs corresponding to the human interleukin 13 receptor alpha chain (IL-13Ralpha). The protein has 76% homology to murine IL-13Ralpha, with 95% amino acid identity in the cytoplasmic domain. Only weak IL-13 binding activity was found in cells transfected with only IL-13Ralpha; however, the combination of both IL-13Ralpha and IL-4Ralpha resulted in substantial binding activity, with a Kd of approximately 400 pM, indicating that both chains are essential components of the IL-13 receptor. Whereas IL-13Ralpha serves as an alternative accessory protein to the common cytokine receptor gamma chain (gammac) for IL-4 signaling, it could not replace the function of gammac in allowing enhanced IL-2 binding activity. Nevertheless, the overall size and length of the cytoplasmic domain of IL-13Ralpha and gammac are similar, and like gammac, IL-13Ralpha is located on chromosome X.