Mhc class I genes of swordtail fishes, Xiphophorus: variation in the number of loci and existence of ancient gene families.

Swordtail fishes and platies in the genus Xiphophorus (order Cyprinodontiformes, Teleostei) encompass 22 closely related species which are the products of a recent adaptive radiation in the streams of Central America. To investigate the evolution of the major histocompatibility complex (Mhc) genes in the period immediately following speciation, the class I genes from 20 of the 22 species were cloned and characterized by sequencing. The analysis revealed the existence of multiple loci (at least seven in some individuals) whose numbers vary among the different species and probably also among individuals of the same species. The variation does not seem to bear any relationship to the taxonomy of the genus. Genes at the different loci are distinguished by their intron sequences and by the presence of characteristic motifs in exons 2 and 3. The variation in copy number of loci may have been effected in part by unequal crossing over occurring between introns of misaligned closely related genes. The sequences of the genes fall into two groups, A and B, which represent ancient lineages. The groups define two families of loci, which diverged from each other an estimated 85 million years ago, before the separation of the Acanthopterygii from the Paracanthopterygii of the advanced bony fishes. Evolution of the genes within each family can be explained by the birth-and-death process driven by gene duplications and mutational differentiation.

Nonlinkage of major histocompatibility complex class I and class II loci in bony fishes.

In tetrapods, the functional (classical) class I and class II B loci of the major histocompatibility complex (Mhc) are tightly linked in a single chromosomal region. In an earlier study, we demonstrated that in the zebrafish, Danio rerio, order Cypriniformes, the two classes are present on different chromosomes. Here, we show that the situation is similar in the stickleback, Gasterosteus aculeatus, order Gasterosteiformes, the common guppy, Poecilia reticulata, order Cyprinodontiformes, and the cichlid fish Oreochromis niloticus, order Perciformes. These data, together with unpublished results from other laboratories suggest that in all Euteleostei, the classical class I and class II B loci are in separate linkage groups, and that in at least some of these taxa, the class II loci are in two different groups. Since Euteleostei are at least as numerous as tetrapods, in approximately one-half of jawed vertebrates, the class I and class II regions are not linked.

Linkage relationships and haplotype polymorphism among cichlid Mhc class II B loci.

The species flocks of cichlid fishes in the Great East African Lakes are paradigms of adaptive radiation and hence, of great interest to evolutionary biologists. Phylogenetic studies of these fishes have, however, been hampered by the lack of suitable polymorphic markers. The genes of the major histocompatibility complex hold the promise to provide, through their extensive polymorphism, a large number of such markers, but their use has been hampered by the complexity of the genetic system and the lack of definition of the individual loci. In this study we take the first substantial step to alleviate this problem. Using a combination of methods, including the typing of single sperm cells, gyno- or androgenetic individuals, and haploid embryos, as well as sequencing of class II B restriction fragments isolated from gels for Southern blots, we identify the previously characterized homology groups as distinct loci. At least 17 polymorphic class II B loci, all of which are presumably transcribed, have been found among the different species studied. Most of these loci are shared across the various cichlid species and genera. The number of loci per haplotype varies from individual to individual, ranging from 1 to 13. A total of 21 distinct haplotypes differing in the number of loci they carry has thus far been identified. All the polymorphic loci are part of the same cluster in which, however, distances between at least some of the loci (as indicated by recombination frequencies) are relatively large. Both the individual loci and the haplotypes can now be used to study phylogenetic relationships among the members of the species flocks and the mode in which speciation occurs during adaptive radiation.

Polymorphism of the HLA class II loci in Siberian populations.

The populations that colonized Siberia diverged from one another in the Paleolithic and evolved in isolation until today. These populations are therefore a rich source of information about the conditions under which the initial divergence of modern humans occurred. In the present study we used the HLA system, first, to investigate the evolution of the human major histocompatibility complex (MHC) itself, and second, to reveal the relationships among Siberian populations. We determined allelic frequencies at five HLA class II loci (DRB1, DQA1, DQB1, DPA1, and DPB1) in seven Siberian populations (Ket, Evenk, Koryak, Chukchi, Nivkh, Udege, and Siberian Eskimo) by the combination of single-stranded conformational polymorphism and DNA sequencing analysis. We then used the gene frequency data to deduce the HLA class II haplotypes and their frequencies. Despite high polymorphism at four of the five loci, no new alleles could be detected. This finding is consistent with a conserved evolution of human class II MHC genes. We found a high number of HLA class II haplotypes in Siberian populations. More haplotypes have been found in Siberia than in any other population. Some of the haplotypes are shared with non-Siberian populations, but most of them are new, and some represent "forbidden" combinations of DQA1 and DQB1 alleles. We suggest that a set of "public" haplotypes was brought to Siberia with the colonizers but that most of the new haplotypes were generated in Siberia by recombination and are part of a haplotype pool that is turning over rapidly. The allelic frequencies at the DRB1 locus divide the Siberian populations into eastern and central Siberian branches; only the former shows a clear genealogical relationship to Amerinds.

Linkage of LMP, TAP, and RING3 with Mhc class I rather than class II genes in the zebrafish.

The LMP2 and LMP7 genes code for subunits of the proteasome, a multimeric enzymatic complex that degrades proteins into peptides. The two subunits replace corresponding constitutively expressed subunits during the immune response. Some of the peptides generated by the proteasome in the cytosol are transported by the products of the TAP1 and TAP2 genes into the lumen of the endoplasmic reticulum and are loaded onto the assembling MHC class I molecules. In mammals, the LMP2, LMP7, TAP1, and TAP2 genes reside in the class II region of the Mhc, closely linked to the RING3 gene. In the present study we identified, cloned, and sequenced the LMP, TAP2, and RING3 genes of the zebrafish, Danio rerio. We identified variants of these genes and used them in a segregation analysis of haploid embryos derived from heterozygous mothers. The analysis revealed that in zebrafish, the LMP2, LMP7, TAP12, and RING3 loci are closely linked but, in contrast to mammals, the LMP/TAP/RING3 cluster resides not in the Mhc class II but in the class I region. We also confirmed that in the zebrafish, the class I and class II regions are not linked to each other. In this species, therefore, the LMP/TAP/RING3 genes are clustered with the class I genes on a chromosome that apparently does not contain any class II genes. The linkage of LMP/TAP/RING3/class I may be the original and the LMP/TAP/RING3/class II a derived arrangement of these genes.

Origin of the North American house mouse.

The house mouse, Mus domesticus, was introduced to the American continent in the post-Columbian era. We have used mouse chromosome 17 DNA probes to trace the origin of the wild house mice on the East Coast of the United States. Of the four probes used, one in particular proved to be informative in this regard. The D17Tu20 probe defines a polymorphism at a locus telomeric of the H-2 complex. TaqI restriction enzyme digests of genomic DNA blotted and hybridized with the D17Tu20 probe revealed the existence of restriction fragments shared by mice from the Atlantic coast of England, France, and the United States but absent in all other tested populations sampled from different parts of the world. This unique polymorphic pattern apparently arose by the loss of two restriction sites in the population on the coast of Brittany. The mutations then presumably spread to England, and from there to the United States. Since the mutations are also present in mice from Florida, English (rather than Spanish) mouse populations may have been either the sole or the main source of immigrants to the eastern United States. This conclusion is also supported by data obtained with the other probes. Presence of the D17Tu20 mutations in some of the laboratory strains indicates that American wild mice contributed to the gene pool of the inbred strains. We postulate that the colonization of North America by English wild mice began in the second half of the seventeenth century.

Class I major histocompatibility complex genes of the red-necked Wallaby, Macropus rufogriseus.

Marsupials are one of three main evolutionary lineages in mammals, the other two being the monotremes and the placental mammals. The marsupial and the placental lineages separated between 120 and 156 million years ago. In this communication, we provide the first molecular description of class I major histocompatibility complex (Mhc) genes in a representative of the marsupial lineage, the red-necked wallaby, Macropus rufogriseus. Three different, nearly full-length class I Mhc sequences were identified in the cDNA library prepared from spleen mRNA of a single wallaby. The three sequences identify at least two loci. Under the assumption that two of the identified sequences are alleles, we designate the three wallaby genes Maru-Mhc-UA*01, Maru-Mhc-UA*02, and Maru-Mhc-UB*01. The three Maru sequences share several codon deletions and insertions not found in the class I genes of placental mammals. Comparisons of genetic distances among the known class I genes suggest that the Maru genes arose from one ancestral element, whereas the class I genes of the placental mammals arose from another, different ancestral element. The absence of an identifiable defect in the three Maru sequences suggests that the genes from which they were derived are functional. Hence, as in placental mammals, there appear to be two functional class I Mhc loci in the marsupials as well.

Trans-species origin of Mhc-DRB polymorphism in the chimpanzee.

Trans-specific evolution of allelic polymorphism at the major histocompatibility complex loci has been demonstrated in a number of species. Estimating the substitution rates and the age of trans-specifically evolving alleles requires detailed information about the alleles in related species. We provide such information for the chimpanzee DRB genes. DNA fragments encompassing exon 2 were amplified in vitro from genomic DNA of ten chimpanzees. The nucleotide sequences were determined and their relationship to the human DRB alleles was evaluated. The alleles were classified according to their position in dendrograms and the presence of lineage-specific motifs. Twenty alleles were found at the expressed loci Patr-DRB1, -DRB3, -DRB4, -DRB5, and at the pseudogenes Patr-DRB6, -DRB7; of these, 13 are new alleles. Two other chimpanzee sequences were classified as members of a new lineage tentatively designated DRBX. Chimpanzee counterparts of HLA-DRB1*01 and *04 were not detected. The number of alleles found at individual loci indicates asymmetrical distribution of polymorphism between humans and chimpanzees. Estimations of intra-lineage divergence times suggest that the lineages are more than 30 million years old. Predictions of major chimpanzee DRB haplotypes are made.

C4 genes of the chimpanzee, gorilla, and orang-utan: evidence for extensive homogenization.

The human complement component 4 is encoded in two genes, C4A and C4B, residing between the class I and class II genes of the major histocompatibility complex. The C4A and C4B molecules differ in their biological activity, the former binding more efficiently to proteins than to carbohydrates while for the latter, the opposite holds true. To shed light on the origin of the C4 genes we isolated cosmid clones bearing the C4 genes of a chimpanzee, a gorilla, and an orang-utan. From the clones, we isolated the fragments coding for the C4d part of the gene (exons and introns) and sequenced them. Altogether we sequenced eight gene fragments: three chimpanzee (Patr-C4-1*01, Patr-C4-1*02, Patr-C4-2*01), two gorilla (Gogo-C4-1*01, Gogo-C4-2*01), and three orang-utan (Popy-C4-1*01, Popy-C4-2*01, Popy-C4-3*01). Comparison of the sequences with each other and with human C4 sequences revealed that in the region believed to be responsible for the functional difference between the C4A and C4B proteins the C4A genes of the different species fell into one group and the C4B genes fell into another. In the rest of the sequence, however, the C4A and C4B genes of each species resembled each other more than they did C4 genes of other species. These results are interpreted as suggesting extensive homogenization (concerted evolution) of the C4 genes in each species, most likely by repeated unequal, homologous, intragenic crossing-over.

One subunit of the transcription factor NF-Y maps close to the major histocompatibility complex in murine and human chromosomes.

The genes coding for the A and B subunits of the transcription factor NF-Y are assigned by a combination of in situ hybridization and analysis of somatic cell hybrids and recombinant mouse strains. NF-YA is assigned to human chromosome 6p21 and to mouse chromosome 17. NF-YB is assigned to human chromosome 12 and to mouse chromosome 10.

Characterization of H-2 congenic strains using DNA markers.

Congenic mouse strains are widely used in mapping traits to specific loci or short chromosomal regions. The precision of the mapping depends on the information available about the length of the differential segment--the segment introduced from the donor into the background strain. Until recently, very few markers flanking the differential locus were known and consequently the length of the foreign segment could only be determined imprecisely. Now, in an attempt to construct a map of the mouse chromosome 17, we have produced a set of DNA markers distributed along the chromosome. These markers provide a new opportunity to measure the length of the differential segment of the congenic strains and thus increase their usefulness for gene mapping. Here we examined the DNA of 96 H-2 congenic strains using 30 DNA markers; of these, the most proximal is located roughly 1.5 centiMorgans (cM) from the centromere and the most distal is about 20 cM telomeric from the H-2 complex (the complex itself being some 20 cM from the centromere). The mapping depends on polymorphism among the input strains and can therefore establish only the minimal length of the differential segment. This point is emphasized by the fact that the average observed length of the differential segment is only about one half of the expected values.

Linkage map of mouse chromosome 17: localization of 27 new DNA markers.

Chromosome 17 of the laboratory variant of the house mouse (Mus musculus L.), MMU17, has been studied extensively, largely because of its involvement in the control of immune response and embryonic as well as male germ cell differentiation. A detailed linkage map of this chromosome is therefore a highly desired goal. As the first step toward achieving this goal, we have isolated, using a LINE 1 repetitive sequence as a probe, 52 anonymous DNA clones from MMU17. Twenty-seven repetitive sequence-free probes isolated from these clones displayed restriction fragment length variation among common inbred strains and could be mapped with the help of recombinant inbred strains, congenic strains, F2 segregants, or intra-t recombinants. Together with markers identified previously, the new markers can be used to construct a map of MMU17 that contains 125 DNA loci. The markers are distributed over a length of approximately 71 cM, which probably represents the entire length of MMU17. Most of the markers reside in the proximal portion of the chromosome, which contains the t and H-2 complexes; this chromosomal region is now fairly well mapped. The distal region of MMU17, on the other hand, is populated by only a few, rather imprecisely mapped markers. Molecular maps are available for most of the H-2 complex and for parts of the t complex.

[Interactions between herbicides and various of the most frequently used drug groups. I. Interactions between chronically administered herbicides: carbendazyme, 2,4-dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid and drugs affecting the circulatory system]. / Interakcje herbicydów i niektórych najczesciej stosowanych grup leków. I. Interakcje herbicydów: Karbendazymu, kwasu 2,4-dichlorofenoksyoctowego i kwasu 2,4,5-trichlorofenoksyoctowego podawanych przewlekle z lekami dzialajacymi na uklad krazenia.

The influence of chronically administered (6 weeks) herbicides (KAR, 2,4-D and 2,4,5-T) on pharmacological effects of quinidine, reserpine and strophantin K in mice and rats was examined in vivo. The investigated herbicides produced in mice an increase in acute toxicity of strophantin K, the acute toxicity of either reserpine oro quinidine being unchanged. The herbicides alone, after 6 weeks administration, significantly increased mean arterial pressure of rats, however the hypotensive response to reserpine was not statistically influenced in these animals as compared with controls. The prolonged administration of investigated herbicides produced also the increased sensibility of heart to the arrhythmic effects of quinidine and ouabain as observed in the ecg pattern in rats treated with increasing doses of these agents. The studies indicate the significance of the possible pharmacological interactions between the drugs affecting cardiovascular system and commonly used herbicides in men.

The effect of labetalol and propranolol on the development of hypertension in spontaneously hypertensive rats.

The effect of long-term administration of propranolol (0.2 and 0.4 mg/kg-1 p.o.) and that of labetalol (2.0, 4.0 and 8.0 mg/kg-1 p.o. and 1 mg/kg-1 i.p.) on the development of hypertension was studied in female rats of SHR/N/Ibn strain. The experiment was started on 6-week-old animals. Rats received the tested drugs once daily between 11 and 12 o'clock. From the 29th day on (i.e. after the age of 10 weeks) the arterial blood pressure was measured bloodlessly twice weekly 2 hours before the daily dose of the drug. Labetalol given p.o. in a dose of 2 mg/kg-1 had no effect on the development of hypertension, while the doses 4.0 and 8.0 mg/kg-1 decreased the arterial pressure by about 4 kPa (30 mm Hg). After drug withdrawal a rapid rise was observed in the arterial pressure to values approaching the values recorded in control animals. In the animals receiving labetalol parenterally (1 mg/kg-1 i.p.) the blood pressure was significantly lower than in control rats. After drug withdrawal the blood pressure increased gradually, however, even on the 54th day of the experiment it was slightly below that in controls. Propranolol given orally in doses of 0.2 mg/kg-1 delayed the development of hypertension, while in the dose of 0.4 mg/kg-1 it prevented its development. After withdrawal of the lower drug dose the blood pressure remained for about 2 weeks at a level similar to the previous one, later on it increased steeply to the values observed in controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Polymorphism of Qa and Tla loci of the mouse.

Congenic lines carrying H-2 haplotypes derived from wild mice were typed serologically with polyclonal and monoclonal antibodies specific for Tla, Qa-1, and Qa-2 antigens. The typing revealed the presence of a minimum of five Tla, four Qa-1, and three Qa-2 alleles in the 32 lines. Two new Tla, two new Qa-1, and one new Qa-2 alleles could be described. This polymorphism of Tla and Qa loci is lower than that detected at the K and D loci in the same lines. The serological typing for Qa-2 antigens correlates remarkably well with previously published results of typing with cytolytic T lymphocytes. This correlation supports the identify of loci detected by the two methods.