Chromosome loss is responsible for segregation at the HPRT locus in Chinese hamster cell hybrids

The phenomenon of segregation of gene expression has been examined in intraspecific somatic cell hybrids. Specifically, segregation at the hypoxanthine guanine phosphoribosyltransferase (HPRT) locus has been studied in hybrids of Chinese hamster cell lines. The role of chromosome segregation, or other chromosomal events has been assessed by detailed comparison of karyotypes in the 6-thioguanine resistant segregants with those of the parental hybrid lines. The results clearly demonstrate that loss of an entire X chromosome is the primary event responsible for segregation at the HPRT locus, while deletion of a portion of the short arm of an X chromosome was also a frequent event. The results provide the first direct evidence for the assignment of the mapping of this locus to the distal region of the short arm. Analysis of chromosome number distributions in the hybrids and segregants suggests that in selecting chromosomal segregants one may also select for hybrid lines with reduced chromosome stability.     

Higher-order genome organization in platypus and chicken sperm and repositioning of sex chromosomes during mammalian evolution

In mammals, chromosomes occupy defined positions in sperm, whereas previous work in chicken showed random chromosome distribution. Monotremes (platypus and echidnas) are the most basal group of living mammals. They have elongated sperm like chicken and a complex sex chromosome system with homology to chicken sex chromosomes. We used platypus and chicken genomic clones to investigate genome organization in sperm. In chicken sperm, about half of the chromosomes investigated are organized non-randomly, whereas in platypus chromosome organization in sperm is almost entirely non-random. The use of genomic clones allowed us to determine chromosome orientation and chromatin compaction in sperm. We found that in both species chromosomes maintain orientation of chromosomes in sperm independent of random or non-random positioning along the sperm nucleus. The distance of loci correlated with the total length of sperm nuclei, suggesting that chromatin extension depends on sperm elongation. In platypus, most sex chromosomes cluster in the posterior region of the sperm nucleus, presumably the result of postmeiotic association of sex chromosomes. Chicken and platypus autosomes sharing homology with the human X chromosome located centrally in both species suggesting that this is the ancestral position. This suggests that in some therian mammals a more anterior position of the X chromosome has evolved independently.     

Syntenic assignments of visual transduction genes in cattle.

To establish syntenic relationships of phototransduction genes, we have mapped the genes encoding the alpha-, beta-, and gamma-subunits of rod cGMP phosphodiesterase (PDE) (PDEA, PDEB, PDEG), the alpha'-subunit of cone PDE (PDEA2), and the rod cGMP-gated channel (CNCG) to bovine syntenic groups. The rod cGMP PDE alpha-, beta-, and gamma-subunit genes map to bovine syntenic groups U22, U15 (chromosome 6), and U21 (chromosome 19), respectively. The rod cGMP-gated channel gene also maps to syntenic group U15, and the bovine cone alpha'-subunit gene maps to U26 (chromosome 26). With the exception of the cone PDE alpha'-subunit gene, which has not been mapped in other mammals, all of these genes have been assigned to conserved chromosomal regions shared among bovine, human, and mouse. A compilation of currently known syntenic assignments and predictions regarding future assignments of phototransduction genes in human, mouse, and cattle is presented.     

Recapitulating the evolution of Afrotheria: 57 genes and rare genomic changes (RGCs) consolidate their history

The decipherment of higher level relationships among the orders of Afrotheria – an extraordinary assumption in mammalian evolution – constitutes one of the major disputes in the evolutionary history of mammals. Recent comprehensive studies of various genomic data, including mitochondrial and nuclear DNA sequences, chromosomal syntenic associations and retroposon insertions support strongly the monophyly of Afrotheria. However, the relationships within Afrotheria have remained ambiguous and there is a necessity for a more sophisticated analysis (i.e. combination of gene phylogeny and Rare Genomic Changes (RGCs)), which could aid in the comprehension of the evolutionary history of this old group of mammals. The present study investigated the phylogenetic relationships within Afrotheria by analysing a data set of coding and non-coding sequences (～32 000 bp) comprising 57 orthologous genes and 31 RGCs, such as chromosomal associations and retroposon insertions, and re-evaluated a molecular timescale for afrotherian mammals using a Bayesian relaxed clock approach. The interordinal afrotherians phylogeny presented here contributed to the elucidation of the evolutionary history of this ancient clade of mammals, which is one of the most unorthodox proposals in mammalian biology. This is critical not only for understanding how Afrotheria evolved in Africa, but also to comprehend the early biogeographical history of placental mammals.     

Disruption and pseudoautosomal localization of the major histocompatibility complex in monotremes

Background

The monotremes, represented by the duck-billed platypus and the echidnas, are the most divergent species within mammals, featuring a flamboyant mix of reptilian, mammalian and specialized characteristics. To understand the evolution of the mammalian major histocompatibility complex (MHC), the analysis of the monotreme genome is vital.

Results

We characterized several MHC containing bacterial artificial chromosome clones from platypus (Ornithorhynchus anatinus) and the short-beaked echidna (Tachyglossus aculeatus) and mapped them onto chromosomes. We discovered that the MHC of monotremes is not contiguous and locates within pseudoautosomal regions of two pairs of their sex chromosomes. The analysis revealed an MHC core region with class I and class II genes on platypus and echidna X3/Y3. Echidna X4/Y4 and platypus Y4/X5 showed synteny to the human distal class III region and beyond. We discovered an intron-containing class I pseudogene on platypus Y4/X5 at a genomic location equivalent to the human HLA-B,C region, suggesting ancestral synteny of the monotreme MHC. Analysis of male meioses from platypus and echidna showed that MHC chromosomes occupy different positions in the meiotic chains of either species.

Conclusion

Molecular and cytogenetic analyses reveal new insights into the evolution of the mammalian MHC and the multiple sex chromosome system of monotremes. In addition, our data establish the first homology link between chicken microchromosomes and the smallest chromosomes in the monotreme karyotype. Our results further suggest that segments of the monotreme MHC that now reside on separate chromosomes must once have been syntenic and that the complex sex chromosome system of monotremes is dynamic and still evolving.     

Gene mapping in marsupials and monotremes. II. Assignments to the X chromosome of dasyurid marsupials

Somatic cell hybrids have been obtained between HPRT Chinese hamster cells and cells from several dasyurid marsupial species. These hybrids show the extensive loss of marsupial chromosomes characteristic of the majority of marsupial-eutherian somatic cell hybrids. Although all of the hybrids expressed the selected marsupial marker, HPRT, the only other markers observed were PGK, GLA, and G6PD, consistent with the conservation of X-linked genes extending to this major group of marsupials. Counterselection confirmed the synteny of PGK and GLA with HPRT, whereas G6PD showed decreased concordance.     

Chicken CD69 and CD94/NKG2-like genes in a chromosomal region syntenic to mammalian natural killer gene complex

In mammals, natural killer (NK) cell C-type lectin receptors were encoded in a gene cluster called natural killer gene complex (NKC). The NKC is not reported in chicken yet. Instead, NK receptor genes were found in the major histocompatibility complex. In this study, two novel chicken C-type lectin-like receptor genes were identified in a region on chromosome 1 that is syntenic to mammalian NKC region. The chromosomal locations were validated with fluorescent in situ hybridization. Based on 3D structure modeling, sequence homology, chromosomal location, and phlylogenetic analysis, one receptor is the orthologue of mammalian cluster of differentiation 69 (CD69), and the other is highly homologous to CD94 and NKG2. Like CD94/NKG2 gene found in teleostean fishes, chicken CD94/NKG2 has the features of both human CD94 and NKG2A. Unlike mammalian NKC, these two chicken C-type lectin receptors are not closely linked but separated by 42 million base pairs according to the chicken draft genome sequence. The arrangement of several other genes that are located outside the mammalian NKC is conserved among chicken, human, and mouse. The chicken NK C-type lectin-like receptors in the NKC syntenic region indicate that this chromosomal region existed before the divergence between mammals and aves. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. The nucleotide sequences have been submitted to the GenBank nucleotide sequence database under the accession number chicken CD69 (DQ156495), CD94/NKG2 (DQ156496), and CD94/NKG2 variant (DQ241793).     

Gene mapping in chicken-Chinese hamster somatic cell hybrids. Serum albumin and phosphoglucomutase-2 structural genes on chicken chromosome 6

Chicken phosphoglucomutase (PGM-2), serum albumin, vitamin D binding protein (Gc) and phosphoribosyl pyrophosphate amidotransferase (PPAT) structural genes have been mapped to chicken chromosome 6 using chicken-Chinese hamster somatic cell hybrids containing this chromosome as the only chicken genetic material. Chicken PGM-2 activity was detected in the hybrids using cellogel electrophoresis and a substrate, ribose-1-phosphate (R-1-P), that allows the detection of PGM-1 activity in mice and PGM-2 activity in humans. Chicken albumin sequences were detected in the hybrids with the use of a labelled chicken serum albumin cloned cDNA. Classical studies have shown linkage of the serum albumin and Gc genes, and the Gc gene also can be localized to chicken chromosome 6. The PPAT gene was localized to this chromosome in previous studies using these hybrids. A homologous linkage group has been identified in mammals and, therefore, a chromosomal linkage group containing at least four genes--Gc, serum albumin, PPAT, and PGM-2--has been conserved over a period of 300 million years, throughout both avian and mammalian evolution.     

Cloning and characterization of the platypus mitochondrial genome

The vertebrate mitochondrial genome is highly conserved in size and gene content. Among the chordates there appears to be one basic gene arrangement, but rearrangements in the mitochondrial gene order of the avian lineages have indicated that the mitochondrial genome may be more variable than once thought. Different gene orders in marsupials and eutherian mammals leave the ancestral mammalian order in some doubt. We have investigated the mitochondrial gene order in the platypus (Ornithorhynchus anatinus), a representative of the third major group of mammals, to determine which mitochondrial gene arrangement is ancestral in mammals. We have found that the platypus mtDNA conforms to the basic chordate gene arrangement, common to fish, amphibians, and eutherian mammals, indicating that this arrangement was the original mammalian arrangement, and that the unusual rearrangements observed in the avians and marsupials are probably lineage-specific. Correspondence to: N.J. Gemmell     

IPCS guidelines for the monitoring of genotoxic effects of carcinogens in humans. International Programme on Chemical Safety

The purpose of these guidelines is to provide concise guidance on the planning, performing and interpretation of studies to monitor groups or individuals exposed to genotoxic agents. Most human carcinogens are genotoxic but not all genotoxic agents have been shown to be carcinogenic in humans. Although the main interest in these studies is due to the association of genotoxicity with carcinogenicity, there is also an inherent interest in monitoring human genotoxicity independently of cancer as an endpoint.The most often studied genotoxicity endpoints have been selected for inclusion in this document and they are structural and numerical chromosomal aberrations assessed using cytogenetic methods (classical chromosomal aberration analysis (CA), fluorescence in situ hybridisation (FISH), micronuclei (MN)); DNA damage (adducts, strand breaks, crosslinking, alkali-labile sites) assessed using bio-chemical/electrophoretic assays or sister chromatid exchanges (SCE); protein adducts; and hypoxanthine-guanine phosphoribosyltransferase (HPRT) mutations. The document does not consider germ cells or gene mutation assays other than HPRT or markers of oxidative stress, which have been applied on a more limited scale.     

Conservation of chromosomes syntenic with avian autosomes in squamate reptiles revealed by comparative chromosome painting

In contrast to mammals, birds exhibit a slow rate of chromosomal evolution. It is not clear whether high chromosome conservation is an evolutionary novelty of birds or was inherited from an earlier avian ancestor. The evolutionary conservatism of macrochromosomes between birds and turtles supports the latter possibility; however, the rate of chromosomal evolution is largely unknown in other sauropsids. In squamates, we previously reported strong conservatism of the chromosomes syntenic with the avian Z, which could reflect a peculiarity of this part of the genome. The chromosome 1 of iguanians and snakes is largely syntenic with chromosomes 3, 5 and 7 of the avian ancestral karyotype. In this project, we used comparative chromosome painting to determine how widely this synteny is conserved across nine families covering most of the main lineages of Squamata. The results suggest that the association of the avian ancestral chromosomes 3, 5 and 7 can be dated back to at least the early Jurassic and could be an ancestral characteristic for Unidentata (Serpentes, Iguania, Anguimorpha, Laterata and Scinciformata). In Squamata chromosome conservatism therefore also holds for the parts of the genome which are homologous to bird autosomes, and following on from this, a slow rate of chromosomal evolution could be a common characteristic of all sauropsids. The large evolutionary stasis in chromosome organization in birds therefore seems to be inherited from their ancestors, and it is particularly striking in comparison with mammals, probably the only major tetrapod lineage with an increased rate of chromosomal rearrangements as a whole.     

Current status of the river buffalo (Bubalus bubalis L.) gene map.

Ninety-nine loci have been assigned to river buffalo chromosomes, 67 of which are coding genes and 32 of which are anonymous DNA segments (microsatellites). Sixty-seven assignments were based on cosegregation of cellular markers in somatic cell hybrids (synteny), whereas 39 were based on in situ hybridization of fixed metaphase chromosomes with labeled DNA probes. Seven loci were assigned by both methods. Of the 67 assignments in somatic cell hybrids, 38 were based on polymerase chain reaction (PCR), 11 on isozyme electrophoresis, 10 on restriction endonuclease digestion of DNA, 4 on immunofluorescence, and 4 on chromosomal identification. A genetic marker or syntenic group has been assigned to each arm of the five submetacentric buffalo chromosomes as well as to the 19 acrocentric autosomes, and the X and Y chromosomes. These same markers map to the 29 cattle autosomes and the X and Y chromosomes, and without exception, cattle markers map to the buffalo chromosome or chromosomal region predicted from chromosome banding similarity.     

Genome Instability in interspecific cell hybrids II. Aminopterin resistance and gene amplification in lines arising from fusions of cells from divergent mammalian species

In order to investigate instances of genetic instability in divergent cell hybrids, we studied several RAT-resistant colonies recovered from fusions between HPRT or TK-deficient rodent cells and marsupial or monotreme cells. Most of these colonies proved to lack HPRT or TK activity and to have survived by acquiring resistance to aminopterin; such aminopterin-resistant lines were never recovered from parent cells subjected to HAT selection. Two of the aminopterin-resistant hybrids over-produced DHFR, and possessed either double minutes or an abnormally banded region, the cytological manifestations of gene amplification. Selection in higher aminopterin concentrations yielded a highly resistant line with 100X wild-type DHFR activity and a large homogeneously staining region. We suggest that interspecific cell hybrids are predisposed to gene amplification and may also show many other types of genetic and chromosomal instability, possibly thein vitro equivalent of the “genomic shock” phenomena described for interstrain or interspecies hybrids of plants or animals. This paper, no. II in a series by these authors, reached the Editorial Office on the date given, although it had been mailed earlier than paper no. III; the latter paper also appears in this number — Eds.     

Cellular retinol-binding protein type II (CRBPII) in adult zebrafish (Danio rerio). cDNA sequence, tissue-specific expression and gene linkage analysis.

We have determined the nucleotide sequence of a zebrafish cDNA clone that codes for a cellular retinol-binding protein type II (CRBPII). Radiation hybrid mapping revealed that the zebrafish and human CRBPII genes are located in syntenic groups. In situ hybridization and emulsion autoradiography localized the CRBPII mRNA to the intestine and the liver of adult zebrafish. CRBPII and intestinal fatty acid binding protein (I-FABP) mRNA was colocalized to the same regions along the anterior-posterior gradient of the zebrafish intestine. Similarly, CRBPII and I-FABP mRNA are colocalized in mammalian and chicken intestine. CRBPII mRNA, but not I-FABP mRNA, was detected in adult zebrafish liver which is in contrast to mammals where liver CRBPII mRNA levels are high during development but rapidly decrease to very low or undetectable levels following birth. CRBPII and I-FABP gene expression appears therefore to be co-ordinately regulated in the zebrafish intestine as has been suggested for mammals and chicken, but CRBPII gene expression is markedly different in the liver of adult zebrafish compared to the livers of mammals. As such, retinol metabolism in zebrafish may differ from that of mammals and require continued production of CRBPII in adult liver. The primary sequence of the coding regions of fish and mammalian CRBPII genes, their relative chromosomal location in syntenic groups and possibly portions of the control regions involved in regulation of CRBPII gene expression in the intestine appear therefore to have been conserved for more than 400 million years.     

Ventilation and metabolic rate in the platypus: insights into the evolution of the mammalian breathing pattern

The platypus (Ornithorhyncus anatinus) is characterized by a rate of oxygen consumption (V(O2))that is higher than that reported for other similar sized monotremes, similar to marsupials and somewhat lower than eutherians. The platypus is also characterized by a breathing pattern, more typical of a diving mammal, with a high 'inspiratory drive' and a post-inspiratory pause. Further, the platypus reveals an attenuated hyperventilatory response to hypoxia and a reduced hyperpnoea to hypercapnia; such a response to these chemical stimuli is commonly observed in semi-fossorial and diving mammals. Nevertheless, under conditions of normoxia, ventilation (V(E))is matched to (V(O(2)) such that the convection requirement (V(E)/V(O2)) is similar to that reported for other mammals (approx. 37). The apparent consistency of the convection requirement in mammals suggests the blueprint for the design of the mammalian respiratory system has remained an interspecies constant in the three divergent extant sub-classes of mammals.     

Evolutionary disruptions of human syntenic groups 3, 12, 14, and 15 in Ateles paniscus chamek (Platyrrhini, primates)

Comparative gene assignments of 18 markers, based on analyses of somatic cell hybrids and previous data in the literature, indicated that human (HSA) syntenic groups 3, 12, 14, and 15 are dissociated in the spider monkey species Ateles paniscus chamek (APC). Markers present in HSA 3p were allocated to APC 3 and APC 9. The HSA 12 cluster was split into two syntenic groups, one mainly including HSA 12p markers in APC 16 and the other, including HSA 12q markers, in APC 2p. The HSA 14q cluster split into three syntenic groups, corresponding to APC 2q, APC 6, and APC 12. Finally, the HSA 15 cluster split into two syntenic groups, APC 2q and APC 3. Comparisons with previous gene assignments and human SROs led to the tentative postulation of rearrangements having occurred during the evolutionary divergence of man and A. paniscus chamek. Chromosome painting data in the congeneric species A. geoffroyi, other New World and Old World primates, and several representative non-primate animals were compared in an attempt to delineate the ancestral and derived conditions underlying the evolutionary rearrangement of syntenic groups in mammals.     

Comparative retinal morphology of the platypus

The purpose of this study is to identify evolutionary origin and fate of anatomic features of the duck‐billed platypus eye. Eyes from the duck‐billed platypus and four key evolutionary basal vertebrates (Pacific hagfish, north hemisphere sea lamprey, and Australian and South American lungfishes) were prepared for light microscopy. In addition to a standard panel of stains, tissues were immunostained against a variety of rod and cone opsins. Finally, published opsin sequences of platypus and several other vertebrate species were aligned and compared with immunohistochemical results. A complete scleral cartilage similar to that seen in birds, reptiles and amphibians encloses the platypus eye. This feature is present in sharks and rays, and in extant relatives of tetrapods, the lungfishes. The choroid lacks a tapetum. The retina is largely avascular and is rod‐dominated, with a minority of red‐ and blue‐ cone immunoreactive photoreceptors. Like marsupials and many nonmammalian vertebrates, cones contain clear inner segment droplets. Double cones were present, a feature not found in eutherian mammals or marsupials. Evaluation of opsins indicates that red and blue immunoreactive cone opsins, but not rhodopsin, are present in the most basal of the extant species examined, the Pacific hagfish. Rhodopsin appears in the Australian and South American lungfishes, establishing emergence of this pigment in an extant relative of tetrapods. Unlike eyes of eutherian mammals, the platypus eye has retained morphologic features present in early tetrapods such as amphibians and their evolutionarily basal sister group, the lungfishes. These include scleral cartilage, double cones and cone droplets. In the platypus, as in other mammals, rod rhodopsin is the predominant photoreceptor pigment, at expense of the cone system. J. Morphol. 2011. © 2011 Wiley‐Liss, Inc.     

Transformation of Hprt gene with sperm DNA

Inactivation of the X chromosome during mammalian spermatogenesis has been postulated to occur by the same mechanism that controls female somatic X chromosome inactivation. We have used DNA-mediated transformation of HPRT- cells to test this idea, because it has been shown previously that inactive X chromosome DNA from somatic cells will not transform HPRT- cells. Isolated DNA from the mature sperm of five mammals (human, mouse, horse, bull, rabbit) were all capable of HPRT transformation, and transformants were confirmed electrophoretically. Measures were taken to ensure that the transformation frequencies observed could not be due to somatic contamination. The positive HPRT transformation result indicates that mature sperm X chromosomal DNA is not modified in the same manner as that of female inactive X chromosomal DNA. Since there is evidence for methylation of the somatic inactive X chromosome, it is possible that methylation, at least for the genes studied, is not involved in sperm X chromosome inactivation.     

Insights into the evolution of mammalian telomerase: Platypus TERT shares similarities with genes of birds and other reptiles and localizes on sex chromosomes

ABSTRACT: BACKGROUND: The TERT gene encodes the catalytic subunit of the telomerase complex and is responsible for maintaining telomere length. Vertebrate telomerase has been studied in placental mammals, fish, and the chicken, but less attention has been paid to other vertebrates. The platypus occupies an important evolutionary position, providing unique insight into the evolution of mammalian genes. We report the cloning of a platypus TERT (pTERT) ortholog, and provide a comparison with genes of other vertebrates. RESULTS: The pTERT encodes a protein with the high homology to marsupial TERT and avian TERT. Like the TERT of sauropsids and marsupials, as well as that of sharks and echinoderms, pTERT contains extended variable linkers in the N-terminal region suggesting that they were present already in basal vertebrates and lost independently in placental mammals and ray-finned fish. Several alternatively spliced pTERT variants structurally similar to avian TERT variants were identified. Telomerase activity is expressed in all platypus tissues similarly to cold-blooded animals and murine rodents. pTERT was localized on pseudoautosomal regions of sex chromosomes X3/Y2, expanding the homology between human chromosome 5 and platypus sex chromosomes. The synteny analysis suggests that TERT co-localized with sex-linked genes in the last common mammalian ancestor. Interestingly, female platypuses express higher levels of telomerase in heart and liver tissues than do males. CONCLUSIONS: pTERT shares many features with TERT of the reptilian outgroup, suggesting that pTERT represents the ancestral mammalian TERT. Features specific to TERT of eutherian mammals have, therefore, evolved more recently after the divergence of monotremes.