Genetic mapping in mammals: chromosome map of domestic cat.

A genetic map of 31 biochemical loci located on 17 feline syntenic (linkage) groups has been derived by somatic cell genetic analysis of cat-rodent hybrids. Most of these syntenic groups have been assigned to one of the 19 feline chromosomes. Comparative linkage analysis of the feline biochemical loci and homologous human loci revealed considerable conservation of linkage associations between the primates and the Felidae (order Carnivora). Many of these same linkage groups have not been conserved in the murine genome. The genetic and evolutionary implications of comparative mapping analysis among mammalian species are discussed.

The human homologs of the raf (mil) oncogene are located on human chromosomes 3 and 4.

Two human genes that are homologous to both the murine transforming gene (oncogene) v-raf and the chicken transforming gene v-mil have been mapped by means of human-rodent somatic cell hybrids to human chromosomes previously devoid of known oncogenes. One gene, c-raf-2, which appears to be a processed pseudogene, is located on chromosome 4. The other gene, c-raf-1, which appears to be the active gene, is located on chromosome 3 and has been regionally mapped by chromosomal in situ hybridization to 3p25. This assignment correlates with specific chromosomal abnormalities associated with certain human malignancies.

Gene for T-cell growth factor: location on human chromosome 4q and feline chromosome B1.

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 promise of comparative genomics in mammals.

Dense genetic maps of human, mouse, and rat genomes that are based on coding genes and on microsatellite and single-nucleotide polymorphism markers have been complemented by precise gene homolog alignment with moderate-resolution maps of livestock, companion animals, and additional mammal species. Comparative genetic assessment expands the utility of these maps in gene discovery, in functional genomics, and in tracking the evolutionary forces that sculpted the genome organization of modern mammalian species.

Genetic characterization of human c-rel sequences.

We isolated and sequenced a human genomic-DNA segment that is homologous to a portion of v-rel, the transforming gene of reticuloendotheliosis virus (strain T). We also localized the human rel sequences to human chromosome 2 by screening a panel of rodent X human somatic-cell hybrids with the newly described human rel segment.

Tyramide signal amplification (TSA)-FISH applied to mapping PCR-labeled probes less than 1 kb in size.

Tyramide signal amplification (TSA)-FISH was used to map one mouse and two human DNA probes of less than 1 kb in size. The two human probes were 319 and 608 bp, and the mouse probe was 855 bp. Probes, made from PCR products, were labeled by incorporating biotin-11-dUTP (human) and biotin-16-dUTP (mouse) during PCR amplification. Signals were readily observed in both interphase and metaphase cells following TSA-FISH for all three genes, whereas conventional FISH experiments produced no signals. The two human ATP-binding cassette (ABC) genes, EST883227 (GenBank Accession No. AA243820) and EST990006 (GenBank Accession No. AA348546), mapped to human chromosomes 7p21 and 17q25. The mouse gene, cmyc (exon 2) mapped to band D2 of mouse chromosome 15. These findings demonstrate the ability of this technique to map small probes (PCR products and expressed sequence tags) of less than 1 kb through highly increased signal amplification.

Identification of a yellow gene-specific protein in Drosophila melanogaster by two-dimensional gel electrophoresis.

Analysis of temperature-sensitive mutants suggests that the yellow (y) gene in Drosophila melanogaster is expressed at a different time in each cell type that gives rise to the various structures of the adult cuticle. An important step in analyzing the regulation of this gene requires identification of the y structural protein. A polypeptide has been identified which correlates with the presence or absence of a functional y gene. Furthermore, this protein has the tissue distribution profile expected of the y structural gene product. The ability to locate this gene was facilitated by the use of coisogenic stocks, two-dimensional electrophoretic protein separation, and an ultrasensitive silver protein stain.

A comparative chromosome banding analysis of the Ursidae and their relationship to other carnivores.

Trypsin G-banded karyotypes of eight species of Ursidae were prepared from retrovirus-transformed skin fibroblast cultures. The banding patterns of all bears are highly conserved, even though their diploid numbers range from 42 to 72. A comprehensive analysis of the homologous banding patterns within the Ursidae and with a hypothesized ancestral carnivore karyotype permitted the reconstruction of three significant chromosomal reorganization events that occurred during the evolution of the modern ursids. The first was a multichromosomal fissioning away from the biarmed (2n = 44) primitive carnivore karyotype, leading to six species of the Ursinae subfamily (2n = 78). The second was a comprehensive chromosome fusion in the lineage that led to the Ailuropodinae (giant panda) subfamily (2n = 44). The third event was a second, independent, but less extensive, centromeric fusion occurring in the line that led to the Tremarctinae (spectacled bear) subfamily (2n = 52). Ursidae karyotypes are not only highly conserved within the family but also exhibit extensive chromosome banding homology with other carnivore families.

Conserved regions of homologous G-banded chromosomes between orders in mammalian evolution: carnivores and primates.

The recent derivation of a biochemical map of 33 loci of the domestic cat (Felis catus) revealed a striking conservation of chromosomal linkage associations between the cat and humans. A comparison of homologous (by linkage criteria) chromosomes by using conventionally extended and high-resolution G-banding of human and feline chromosomes is presented. Four criteria for establishing probable cytogenetic homologies of chromosomal regions were invoked: (i) map placement of homologous genes to the same chromosomes; (ii) cytological correlation of G-banding pattern; (iii) placement of homologous genes, by regional gene mapping, in the region of cytological homology; and (iv) a requirement that the putative region of homology be ancestral and evolutionarily conserved within their respective orders. Five subchromosomal regions (homologous to human chromosome 1p, 2p, 2q, 12, and X) were found to be conserved and homologous by all the stated criteria. The conserved regions constitute nearly 20% by length of the human chromosomal genome. The implications of conservation of chromosome homologies between mammalian orders whose last common ancestor became extinct more than 60 million years ago is discussed.

Gene networks in development.

A model is presented for the formation of temporal and spatial patterns of cell types during the development of organisms. It is demonstrated that very simple random networks of interactions among genes that affect expression may lead to the autonomous development of patterns of cell types. It is required that the networks contain active feedback loops and that there is limited communication among cells. The only elements of the model, gene interactions, are specified by the DNA nucleotide sequences of the genes. Therefore, the model readily explains how the control of development is specified by the organism's DNA. In the context of this model, the formation of positional information and its interpretation becomes a single process.

Chromosomal mapping of enzyme loci in the domestic cat: GSR to C2, ADA and ITPA to A3, and LDHA-ACP2 to D1.

A panel of 42 rodent X cat somatic cell hybrids segregating individual cat chromosomes in different combinations was used to assign five isozyme structural loci to cat chromosomes. The feline homolog for glutathione reductase (GSR) was mapped to chromosome C2. Adenosine deaminase (ADA) and inosine triphosphatase (ITPA) were located on chromosome A3. Lactate dehydrogenase-A (LDHA) and acid phosphatase-2 (ACP2) were reassigned to chromosome D1. Localization of these genes increases the known feline genetic map and extends the known syntenic homologies between the cat and other mammalian species.

Chromosomal evolution of the Canidae. I. Species with high diploid numbers.

The Giemsa banding patterns of seven canid species, including the grey wolf (Canis lupus), the maned wolf (Chrysocyon brachyurus), the bush dog (Speothos venaticus), the crab-eating fox (Cerdocyon thous), the grey fox (Urocyon cinereoargenteus), the bat-eared fox (Otocyon megalotis), and the fennec (Fennecus zerda), are presented and compared. Relative to other members of Canidae, these species have high diploid complements (2n greater than 64) consisting of largely acrocentric chromosomes. They show a considerable degree of chromosome homoeology, but relative to the grey wolf, each species is either missing chromosomes or has unique chromosomal additions and rearrangements. Differences in chromosome morphology among the seven species were used to reconstruct their phylogenetic history. The results suggest that the South American canids are closely related to each other and are derived from a wolf-like progenitor. The fennec and the bat-eared fox seem to be recent derivatives of a lineage that branched early from the wolf-like canids and which also includes the grey fox.

Chromosomal evolution of the Canidae. II. Divergence from the primitive carnivore karyotype.

The Giemsa-banding patterns of chromosomes from the arctic fox (Alopex lagopus), the red fox (Vulpes vulpes), the kit fox (Vulpes macrotis), and the raccoon dog (Nyctereutes procyonoides) are compared. Despite their traditional placement in different genera, the arctic fox and the kit fox have an identical chromosome morphology and G-banding pattern. The red fox has extensive chromosome arm homoeology with these two species, but has only two entire chromosomes in common. All three species share some chromosomes with the raccoon dog, as does the high diploid-numbered grey wolf (Canis lupus, 2n = 78). Moreover, some chromosomes of the raccoon dog show partial or complete homoeology with metacentric feline chromosomes which suggests that these are primitive canid chromosomes. We present the history of chromosomal rearrangements within the Canidae family based on the assumption that a metacentric-dominated karyotype is primitive for the group.

ERV3, a full-length human endogenous provirus: chromosomal localization and evolutionary relationships.

A full-length human endogenous provirus termed ERV3 was isolated from a human fetal recombinant DNA library by low stringency hybridization with two probes: baboon endogenous virus LTR; and a pol-env subclone from the endogenous chimpanzee provirus, CH2. DNA sequencing within the clone and comparisons with other retroviruses revealed that ERV3 contains gag and pol gene sequences that are significantly related to those of mammalian type C retroviruses and previously described human endogenous proviruses. The ERV3 genome was determined to reside at a single locus on human chromosome 7 using a panel of rodent X human somatic cell hybrids.

Genetic mapping of endogenous RD-114 retroviral sequences of domestic cats.

The RD-114 family of endogenous retroviral sequences in domestic cats has been shown to consist of approximately 20 copies of genetically divergent virogenes per haploid genome. The chromosomal localization for four endogenous sequences (RDV1-4) was accomplished by correlating the occurrence of specific feline chromosomes with diagnostic viral DNA fragments in a panel of cat X rodent somatic cell hybrids. Analysis of the hybrid panel revealed that endogenous RD-114 sequences are dispersed on multiple cat chromosomes, that certain proviral segments are polymorphic with respect to the presence or absence of virus, and that a restriction fragment characteristic of inducible RD-114 resides on a single feline chromosome (B3), probably at a single locus.

Cytogenetic methodologies for gene mapping and comparative analyses in mammalian cell culture systems.

Presented here are the detailed methods employed in our laboratory for gene mapping and cytogenetic analyses in human beings, in the domestic cat, and in other mammalian species. Induced in the procedures are: 1) establishment of primary fibroblast and lymphoid cell cultures; 2) heterologous cell fusion for production of rapidly proliferating cell hybrids; 3) cellular transformation of primary fibroblasts using an oncogenic retrovirus; 4) cell synchronization for high-resolution banding of promethaphase chromosomes; 5) chromosome-banding procedures, including G-banding, alkaline G-11, and Q-banding; and 6) in situ hybridization of radiolabeled molecular clones to metaphase chromosomes for regional gene localization.

Dynamic and nonspecific dispersal of human T-cell leukemia/lymphoma virus type-I integration in cultured lymphoma cells.

The progression of HTLV-I proviral integration over a 3-year period of in vitro culture was examined in two human lymphoma lines, Hut 102 and MJ. Using specific HTLV-I molecular clones and a Southern analysis at different cell passages, Hut 102 increased from 2 to 19 integrated proviral integrations while MJ increased to at least 25 different integrations by passage 43. During the progress of increased superinfection and novel integration in vitro some of the previous proviral integrations were lost from the cultures. The 19 integrations of late passage Hut 102 cells were shown to be dispersed to 19 different human chromosomes by analysis of 34 distinct rodent X Hut 102 somatic cell hybrids which segregated human chromosomes (and included proviral integrations) in different combinations. The two primary integrations in Hut 102 were located on human chromosomes 4 and 20, respectively. A similar pattern of nonspecific integration was observed in somatic cell hybrid analysis of the 25 proviral integrations of MJ. The dynamic infection-reintegration process in vitro revealed in these studies may confuse experimental verification of potential cis acting functions of HTLV-I in the as yet poorly understood mechanism of neoplastic transformation.

Chromosomal organization of the human dihydrofolate reductase genes: dispersion, selective amplification, and a novel form of polymorphism.

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.

Organization and chromosomal specificity of autosomal homologs of human Y chromosome repeated DNA.

The human Y chromosome contains a group of repeated DNA elements, identified as 3.4-kilobase pair (kb) fragments in Hae III digests of male genomic DNA, which contain both Y-specific and non-Y-specific sequences. We have used these 3.4-kb Hae III Y fragments to explore the organizational properties and chromosomal distribution of the autosomal homologs of the non-Y-specific (NYS) 3.4-kb Hae III Y elements. Three distinct organizations, termed domains, have been identified and shown to have major concentrations on separate chromosomes. We have established that domain K is located on chromosome 15 and domain D on chromosome 16 and suggested that domain R is on chromosome 1. Our findings suggest that each domain is composed of a tandemly arrayed cluster of a regularly repeating unit containing two sets of repeated sequences: one that is homologous to the NYS 3.4-kb Hae III Y sequences and one that does not cross-react with the 3.4-kb Hae III Y repeats. Thus, these autosomal repeated DNA domains, like their Y chromosome counterparts, consist of a complex mixture of repeated DNA elements interspersed among each other in ways that lead to defined periodicities. Although each of the three identified autosomal domains cross-reacts with 3.4-kb Hae III Y fragments purified from genomic DNA, the length periodicities and sequence content of the autosomal domains are chromosome specific. The organizational properties and chromosomal distribution of these NYS 3.4-kb Hae III homologs seem inconsistent with stochastic mechanisms of sequence diffusion between chromosomes.