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.

Human endothelial cell growth factor: cloning, nucleotide sequence, and chromosome localization.

Several of the endothelial cell polypeptide mitogens that have been described probably play a role in blood vessel homeostasis. Two overlapping complementary DNA clones encoding human endothelial cell growth factor (ECGF) were isolated from a human brain stem complementary DNA library. Southern blot analysis suggested that there is a single copy of the ECGF gene and that it maps to human chromosome 5 at bands 5q31.3 to 33.2 A 4.8-kilobase messenger RNA was present in human brain stem messenger RNA. The complete amino acid sequence of human ECGF was deduced from the nucleic acid sequence of these clones; it encompasses all the well-characterized acidic endothelial cell polypeptide mitogens described by several laboratories. The ECGF-encoding open reading frame is flanked by translation stop codons and provides no signal peptide or internal hydrophobic domain for the secretion of ECGF. This property is shared by human interleukin-1, which is approximately 30 percent homologous to ECGF.

The human erg gene maps to chromosome 21, band q22: relationship to the 8; 21 translocation of acute myelogenous leukemia.

There is accumulating evidence to support that genes on chromosome 21 play an important role in the development of pathologies associated with leukemia, Down's syndrome, and Alzheimer's disease. We have previously described erg, a human gene related to the ets oncogene. In this study, we have regionally assigned the erg gene to chromosome 21q22.3 by using somatic cell hybrids and in situ hybridization analysis. In light of this chromosome assignment, the relationship of erg to the 21q translocation breakpoint characteristic of acute myelogenous leukemia (AML) was considered. By using a DNA probe that is specific for the erg gene, a panel of rodent-human cell hybrids was analyzed by the Southern technique to study specific chromosome translocations occurring in acute myeloblastic leukemia. The erg gene was found to translocate from chromosome 21 to 8 in the t(8; 21) (q22; q22), a non-random translocation found in patients with acute myelogenous leukemia of the subgroup M2 (AML-M2). The localization of the erg gene to chromosome 21q22 raises the possibility that this gene may be involved in the pathogenesis of AML-M2.

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.

Clinical and molecular analyses of deletion 3p25-pter syndrome.

Hemizygous deletion of 3p25-pter is associated with a phenotype of profound growth failure, microcephaly, characteristic facial changes, and mental retardation. Since the severity may be quite variable, we have studied 3 cases of del 3p25-pter to define the clinical manifestations and the critical chromosome region for phenotypic expression. The patient we now report died at age 6 months and provided an opportunity for a detailed necropsy analysis for only the second time in a del(3p) patient. He had marked hypoplasia of all organs, hypomyelination of white matter, and multiple renal cortical microcysts. Ordered genomic markers from the distal regions of chromosome 3p aided in determining the parent of origin of each deletion and in defining the boundaries of the deleted chromosomal segments. The deleted markers distal to the RAF1 oncogene in 2 of the 3 patients were consistently hemizygous. One patient had an interstitial deletion based on evidence of diploid inheritance of one of the most distal loci (D3S17). Available genetic linkage maps suggest that the deletion spans at least 19 centimorgans (cM).

Characterization and chromosomal localization of a new protein disulfide isomerase, PDIp, highly expressed in human pancreas.

Protein disulfide isomerase (PDI) catalyzes protein folding and thiol-disulfide interchange reactions. The enzyme is localized in the lumen of endoplasmic reticulum (ER) and is abundant in secretory cells of various tissues. In this study we describe the isolation and characterization from human pancreas of a new protein, PDIp, that is structurally and functionally related to PDIs. PDIp cDNA is 1,659 bp in length and predicts a protein with an open reading frame of 511 amino acids. PDIp amino acid sequence shows 46% identity and 66% similarity to that of human PDI. PDIp possesses two thioredoxin-like active sites (WCGHCQ and WCTHCK) and an endoplasmic reticulum retention signal sequence, KEEL, at the carboxyl terminus. Northern analysis of normal human tissues and various human tumor cell lines revealed PDIp mRNA (2.0 kb) expression only in the normal pancreas. Recombinant PDIp protein catalyzed reductive cleavage of insulin and renaturation of reduced RNaseA. Somatic cell genetics and fluorescence in situ hybridization localized the PDIp gene to the short arm of human chromosome 16. It is concluded that PDIp is a new member of the PDI family and is highly expressed in human pancreas.

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.

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.

Comparative analyses of heterochromatin in Microtus: sequence heterogeneity and localized expansion and contraction of satellite DNA arrays.

Southern blotting, C-banding, base-specific fluorochrome staining, and fluorescence in situ hybridization were used to analyze the constitutive heterochromatin in eight species and subspecies of arvicolid rodents (genus Microtus). Autosomal centromeric regions portrayed considerable variability between species in the amount of C-band-positive material present; e.g., M. chrotorrhinus showed relatively little, whereas M. cabrerae exhibited extensive centromeric staining. Autosomal interstitial C-bands were noted in M. guentheri and two subspecies of M. ochrogaster. All Y chromosomes examined were predominately or completely heterochromatic, as were substantial portions of the giant X chromosomes in three species (M. agrestis, M. cabrerae, and M. chrotorrhinus). Hoechst 33258 staining (with its affinity for AT binding sites) showed bright fluorescence on the heterochromatin of the sex chromosomes of M. agrestis and moderate fluorescence on those of M. cabrerae and M. chrotorrhinus; however, only the heterochromatin of M. cabrerae and M. chrotorrhinus hybridized with an AT-rich satellite DNA probe (MSAT-160) isolated from M. chrotorrhinus. Hoechst 33258-bright autosomal centromeres of M. arvalis and M. cabrerae also hybridized to the probe, whereas the Hoechst 33258-bright Y chromosomes of M. arvalis and M. guentheri did not. Two pairs of autosomes in M. guentheri are comprised of six distinct regions, based upon C-banding, Hoechst 33258 staining, chromomycin A3/distamycin A staining, and in situ hybridization. The centromeric regions of acrocentric autosomes known to retain conserved G-banding patterns may exhibit variable hybridization intensity when different species or subspecies are compared. M. ochrogaster portrays considerable intersubspecific variability in the size and location of autosomal telomeric and interstitial C-bands that are also sites of hybridization. These latter two findings illustrate that dramatic differences in copy number of the tandem satellite array can exist at homologous chromosomal positions both within and between species.

Rapid, localized amplification of a unique satellite DNA family in the rodent Microtus chrotorrhinus.

A novel satellite DNA family (called MSAT-2570) was isolated and characterized from the rodent Microtus chrotorrhinus. With a length of 2,570 bp the repeat unit is among the largest yet reported in mammals and comprises a series of short direct and inverted repeats. These repeat motifs may prevent nucleosome formation or represent an endless source of genetic variation. Restriction enzyme digestion using the two pairs of isoschizomers HpaII/MspI and MboI/Sau3AI demonstrated tissue specific differences in satellite DNA methylation that may reflect variable chromatin conformation or differences in patterns of gene expression. The sex chromosomes of M. chrotorrhinus are usually large in size among mammals, comprising 15%-20% of the karyotype and containing large blocks of heterochromatin. In situ hybridization of the satellite DNA revealed chromosomal localization predominantly to sex chromosome heterochromatin. A survey of related rodents including three congeneric species also with giant sized sex chromosomes demonstrated that MSAT-2570 is present only in the genome of M. chrotorrhinus. However, another previously reported satellite DNA also isolated from M. chrotorrhinus has been shown to reside on sex chromosome heterochromatin in one of the other three species, indicating that these giant blocks of heterochromatin are complex in structure and comprise multiple, unrelated satellite DNA families.

Heterogeneity in the concerted evolution process of a tandem satellite array in meadow mice (Microtus).

The evolutionary history of a 160-bp tandem satellite array, originally described from Microtus chrotorrhinus and called MSAT-160, was examined in related species of arvicolid rodents by sequence analyses, quantitative dot blotting, and Southern blotting. Results indicate that MSAT-160 is present in 12 of the 20 species and subspecies of Microtus assayed, but not in species belonging to any of the eight other genera examined. DNA from each species containing MSAT-160 was digested with 12 restriction endonucleases and restriction patterns were obtained reflecting the variable extent of homogenization of any given variant in different species. For example, with MboI digestion, M. chrotorrhinus produced a type A ladder pattern where most monomers contain the restriction site, M. ochrogaster generated a type B pattern where most monomers lack the site, and M. agrestis yielded a pattern intermediate between the A and B types. Further, dot blotting revealed copy-number differences between species. These findings indicate that changes in the periodic structure and amount of satellite DNA have occurred since these species last shared a common ancestor. In addition, various species-specific patterns were documented, illustrating that mechanisms other than genome-wide homogenization, such as stochastic mutation, out-of-register crossing over, deletion, and random amplification also play a role in structuring tandem arrays. Stochastic mutation and homogenization rates in satellite DNA, levels of species diversity, and magnitudes of chromosomal divergence differ significantly in Microtus, Mus and Ctenomys, the three rodent lineages examined.

Phylogenetic history of LINE-1 among arvicolid rodents.

The extent of restriction fragment sharing among arvicolid rodents was examined following Southern blotting with a reverse transcriptase region probe from long interspersed nuclear element 1 (LINE-1 or L1). DNAs from 30 species belonging to nine genera were digested with 11 restriction endonucleases. Following hybridization discrete bands were scored with respect to their presence or absence and intensity, and both within- and between-species comparisons were conducted. Intraspecific analyses revealed low but detectable levels of variation. Interspecific comparisons revealed three groups of bands: those present in all 30 species (6 out of a total of 248 bands), those phylogenetically informative in two or more species (130 out of 248), and those unique to a single species (114 out of 248). A multistate data matrix consisting of species by codes representing the intensities of informative bands was analyzed by maximum parsimony. Further, distance values between species were converted to rates using estimated fossil divergence times. Both the parsimony and rate analyses revealed differences between species in the extent of band sharing and in the intensities of common bands, indicating that the amplification and movement of LINE elements has occurred in episodic bursts during the history of this group. Systematic interpretations of the evolutionary trees were concordant with those previously obtained using other data sets, suggesting that although the amplification of repetitive sequences may occur episodically in this taxonomic group, there do appear to be some constraints.

Nucleotide sequence and genomic organization of a tandem satellite array from the rock vole Microtus chrotorrhinus (Rodentia).

A tandem satellite array (herein named MSAT-160) has been isolated and characterized from the rodent Microtus chrotorrhinus. Sequence data from 15 partial or complete monomers revealed a repeat unit length of 160 bp. This unit length was apparently derived from two shorter sub-motifs, one a tetramer (GAAA), the other a hexamer (CTTTCT), through polymerase slippage and mutation. Collectively, perfect or imperfect variants of these two motifs comprise nearly 60% of the component. Southern blot analyses of genomic DNA digested with 14 different restriction endonucleases indicated that most enzymes yielded either classical type A or type B restriction patterns, while RsaI yielded a pattern that combined features of both the A and B types, and BamHI appeared to lack sites altogether in MSAT-160. An examination of restriction patterns from 16 individuals with three enzymes failed to identify intraspecific variation, while a related study compared 11 species and documented interspecific distinctiveness (Modi, submitted). Fluorescence in situ hybridization indicated that the satellite DNA was located at the centromeres of several autosomes and at sex chromosome heterochromatin.

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.

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.

Molecular characterization of the complex sex-chromosome heterochromatin in the rodent Microtus chrotorrhinus.

The sex chromosomes of Microtus chrotorrhinus are unusually large compared to those of other mammals, comprising about 20% of the karyotype and containing substantial amounts of constitutive heterochromatin. Previous studies have described two highly repeated DNA families (MSAT-160 and MSAT-2570) that localize to this heterochromatin (Modi, 1992, 1993c). The present report describes a third satellite DNA family (termed MSAT-21) in M. chrotorrhinus that is also located in the sex heterochromatin. This repeat consists of diverged copies (average similarity = 75%) of a tandemly repeated 21-mer. Southern blotting of MSAT-21 revealed that although some higher order (5-20 kb) repeats do exist, none has spread throughout an appreciable portion of the genome. Pulsed field gel experiments indicated that most of the larger arrays (50-700 kb) of all three satellite families are distributed across numerous size classes, suggesting that the three repeats are interspersed with one another in this heterochromatin. Analysis of a boundary between MSAT-21 and MSAT-160 showed that the junction monomers of each satellite are intact and that a pentanucleotide has apparently been transferred from MSAT-21 to MSAT-160 via recombination. Sequence comparisons of MSAT-160 with another rodent satellite and with the U3 region of the Rous sarcoma virus (RSV) long terminal repeat identified inverted repeats and similarities with viral enhancer domains in the rodent sequences. Additionally, the MSAT-21 consensus was found to be similar to the R region of RSV, suggesting a retroviral ancestor for these rodent repeated DNA families.

Mapping the human pulmonary surfactant-associated protein B gene (SFTP3) to chromosome 2p12-->p11.2.

Pulmonary surfactant, a lipoprotein complex is essential for normal lung function. The non-serum surfactant-associated proteins, SP-A, SP-B, and SP-C, play important roles in the biology of pulmonary surfactant. We have mapped the human SP-B gene (SFTP3) to chromosome 2, band 2p12-->p11.2 by fluorescent in situ hybridization.

Confirmed assignment of a novel human tyrosine kinase gene (JAK1A) to 1p32.3-->p31.3 by nonisotopic in situ hybridization.

JAK1A is a recently isolated class 3 (nonreceptor) tyrosine kinase that has catalytic domain sequence homology with other kinases and is known to be ubiquitously expressed in all human tissues thus far examined. The gene for this enzyme had previously been localized to chromosome 1 using somatic cell hybrids and linkage analyses. In the present study, fluorescence in situ hybridization was utilized to confirm its localization and regionally assign the gene to chromosome region 1p32.3-->p31.3.

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.

Evolutionary histories of highly repeated DNA families among the Artiodactyla (Mammalia).

Six highly repeated DNA families were analyzed using Southern blotting and fluorescence in situ hybridization in a comparative study of 46 species of artiodactyls belonging to seven of the eight extant taxonomic families. Two of the repeats, the dispersed bovine-Pst family and the localized 1.715 component, were found to have the broadest taxonomic distributions, being present in all pecoran ruminants (Giraffidae, Cervidae, Antilocapridae, and Bovidae), indicating that these repeats may be 25-40 million years old. Different 1.715 restriction patterns were observed in different taxonomic families, indicating that independent concerted evolution events have homogenized different motifs in different lineages. The other four satellite arrays were restricted to the Bovini and sometimes to the related Boselaphini and Tragelaphini. Results reveal that among the two compound satellites studied, the two components of the 1.711a originated simultaneously, whereas the two components of the 1.711b originated at two different historical times, perhaps as many as 15 million years apart. Systematic conclusions support the monophyly of the infraorder Pecora, the monophyly of the subfamily Bovinae (containing the Boselaphini, Bovini, and Tragelaphini), an inability to resolve any interrelationships among the other tribes of bovids, paraphyly of the genus Bos with respect to Bison, and a lack of molecular variation among two morphologically and ecologically distinct subspecies of African buffaloes (Syncerus caffer cafer and S. c. nanus). Cytogenetically, a reduction in diploid chromosome numbers through centric fusion in derived karyotypes is accompanied by a loss of centromeric satellite DNA. The nilgai karyotype contains an apparent dicentric chromosome as evidenced by the sites of 1.715 hybridization. Telomeric sequences have been translocated to the centromeres without concomitant chromosomal rearrangement in Thompson's gazelle.