Evolution of the neuropeptide Y receptor family: gene and chromosome duplications deduced from the cloning and mapping of the five receptor subtype genes in pig.

Neuropeptide Y (NPY) receptors mediate a variety of physiological responses including feeding and vasoconstriction. To investigate the evolutionary events that have generated this receptor family, we have sequenced and determined the chromosomal localizations of all five presently known mammalian NPY receptor subtype genes in the domestic pig, Sus scrofa (SSC). The orthologs of the Y(1) and Y(2) subtypes display high amino acid sequence identities between pig, human, and mouse (92%-94%), whereas the Y(4), Y(5), and y(6) subtypes display lower identities (76%-87%). The lower identity of Y(5) is due to high sequence divergence in the large third intracellular loop. The NPY1R, NPY2R, and NPY5R receptor genes were localized to SSC8, the NPY4R to SSC14, and NPY6R to SSC2. Our comparisons strongly suggest that the tight cluster of NPY1R, NPY2R, and NPY5R on human chromosome 4 (HSA4) represents the ancestral configuration, whereas the porcine cluster has been split by two inversions on SSC8. These 3 genes, along with adjacent genes from 14 other gene families, form a cluster on HSA4 with extensive similarities to a cluster on HSA5, where NPY6R and >13 other paralogs reside, as well as another large cluster on HSA10 that includes NPY4R. Thus, these gene families have expanded through large-scale duplications. The sequence comparisons show that the NPY receptor triplet NPY1R-NPY2R-NPY5R existed before these large-scale duplications.

Gene duplications in early metazoan evolution.

Major increases in complexity during animal evolution occurred at the transition from a unicellular protozoan to a multicellular metazoan, the evolution of Bilateria from diploblasts (possibly the Cambrian explosion) and during early vertebrate evolution. A role for gene duplication in the third event has been widely discussed. Here I examine the possible role of gene duplications and domain shuffling in the first two events. There is evidence for a wave of gene duplications and shuffling which may have paved the way for multicellularity; there are also examples of gene duplications that may have facilitated the transition from diploblasts to Bilateria.

Lampetra fluviatilis neurotrophin homolog, descendant of a neurotrophin ancestor, discloses the early molecular evolution of neurotrophins in the vertebrate subphylum.

We have isolated a neurotrophin from the lamprey that permitted us to perform a phylogenetic analysis of the neurotrophin gene family that dates back more than 460 million years to the early vertebrate ancestors. The results show that the neurotrophin gene family was originally formed by two subsequent duplications. The duplication that formed nerve growth factor, neurotrophin-3, brain-derived neurotrophic factor, and neurotrophin-4/5 occurred after the split of lampreys but before the split of cartilaginous fish from the main vertebrate lineage. Compilation of chromosomal gene maps around the neurotrophins shows that they are located in paralogous regions, suggesting that the genes were formed at major duplication events possibly by complete genome doubling. Analysis of two isolated Trk receptor sequences shows similar results as for the lamprey neurotrophin. Multiple neurotrophin and Trk genes, including neurotrophin-6 and -7, have been found in bony fish, and we suggest that the extra genes were formed by an additional duplication in the bony fish lineage. Analysis of lamprey Trk mRNA expression in the adult brain shows that the genes are expressed in all regions analyzed so far. Together, the results suggest that the duplications of ancestral neurotrophin and Trk genes at an early vertebrate stage have permitted evolution to bring about differential neurotrophin and Trk expression, thereby allowing the formation of specific functions in selective neuronal populations.

Evolution of the vertebrate genome as reflected in paralogous chromosomal regions in man and the house mouse.

Gene constellations on several human chromosomes are interpreted as indications of large regional duplications that took place during evolution of the vertebrate genome. Four groups of paralogous chromosomal regions in man and the house mouse are suggested and are believed to be conserved remnants of the two or three rounds of tetraploidization that are likely to have occurred during evolution of the vertebrates. The phenomenon of differential silencing of genes is described. The importance of conservation of linkage of particular genes is discussed in relation to genetic regulation and cell differentiation.

Lysosomal mutations increase susceptibility to anaesthetics.

The anaesthetic responses of homozygous mutant mice were compared with those of their normal heterozygous littermates. The two recessive mutations studied were beige (bg) and reduced pigmentation (rp). Homozygosity for either significantly increased the sleeping time of both sexes after treatment with pentobarbital, tribromoethanol or the steroid anaesthetic alphaxalone.

Genetic variation of peptidase and pyrophosphatase in the chicken.

Differences in electrophoretic mobilities of two chicken liver, kidney, and spleen enzymes have been demonstrated. A peptidase cleaving the dipeptide L-leucyl-alanine was found to be under the control of a single locus, Pep-1, in crosses between two breeds of chicken, White Leghorn (WL) and Rhode Island Red (RIR). Three alleles, a, b, and c were segregating in the WL breed but only two of these, a and c, seemed to be present in the RIR stock. The other enzyme investigated here was pyrophosphatase, and was shown to be under the control of one locus, Pyp, with two alleles, a and b. The two alleles had similar frequencies in the RIR breed, whereas in the WL breed, the a allele was more frequent.

A gene (Bmn) controlling beta-mannosidase activity in the mouse is located in the distal part of chromosome 3.

A gene (Bmn) with a major effect on beta-mannosidase activity in kidney and liver of the house mouse was revealed by assay with the synthetic substrate p-nitrophenyl-beta-D-mannoside. Activity is low in DBA/2J and CSB mice and high in C57BL/6J mice. By the use of the BXD series of recombinant inbred strains and by crosses between C57BL and CSB, it was possible to map the gene to the distal part of chromosome 3 by demonstration of linkage to a gene for cadmium resistance, cdm, as well as to the Adh-3 locus.

Interferon-regulated influenza virus resistance gene Mx is localized on mouse chromosome 16.

Genomic Southern blots of mouse-hamster somatic cell hybrids were analyzed with a probe prepared from a cDNA encoding murine Mx protein, the product of the interferon-regulated influenza virus resistance allele Mx+. Results of this analysis indicate that the Mx gene is located on mouse chromosome 16. In appropriate backcross mice, no linkage was observed between Mx and md, a marker previously mapped close to the centromere of chromosome 16, suggesting a more distal localization of Mx.

Morphine metabolism in mouse brain.

Morphine UDP-glucuronyltransferase activity was demonstrated in the brain of mice from recombinant inbred strains of the BXD series. The formation rate of morphine-3-glucuronide was about 4 fold higher in the progenitor DBA as compared to the C57BL strain.

Localization of the gene for the vitamin B12 binding protein, transcobalamin II, near the centromere on mouse chromosome 11, linked with the hemoglobin alpha-chain locus.

Somatic cell hybrids, recombinant inbred (RI) mouse strains, and backcross breeding experiments were used to locate the gene of transcobalamin II (Tcn-2), the vitamin B12 binding protein in mouse serum. TCN-2 was found to be useful genetic marker in the somatic cell hybrids. Selected hybrid clones were derived from fusions between GR mouse cells and the Chinese hamster cell line E36. Analysis of mouse specific chromosomal enzyme markers in relationship to TCN-2 secretion, in the hybrid clones, provided provisional evidence for assignment of the Tcn-2 locus to chromosome 11. The strain distribution pattern of the TCN-2 variants S and F in the RI series CXS, constructed from the cross of BALB/cHeA (TCN-2S) with STS/A (TCN-2F), implied a close linkage with the hemoglobin alpha-chain locus (Hba) on chromosome 11. Backcross breeding using inbred strains confirmed these findings and located the Tcn-2 gene closest to the centromere, linked with waved 2 (wa-2) and Hba with recombination frequencies of 6.9 and 19.2% each. The linkage group Tcn-2/wa-2/Hba was established.

Teratogenicity of 2,3,7,8-tetrachlorodibenzofuran in BXD recombinant inbred strains.

A series of recombinant inbred strains called BXD [produced from a cross between C57BL/6J (B6) and DBA/2J (D2)] were given single i.p. doses of 0.6 mg/kg 2,3,7, 8-tetrachlorodibenzofuran (TCDBF) on day 12 of gestation. The uteri were examined in late gestation with respect to resorptions and fetal death, and fetal malformations. The strains of the B6-type with respect to Ah-locus (Nos. 5, 6, 8, 11, 12, 14, 16 and 29) that are Ah-responsive, exhibited cleft palates in 80-100% of all fetuses, while hydronephrosis occurred at a rate of 20-70%. These two types of malformation are well recognized from earlier experiments with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and its structural analogues, including TCDBF. In the strains of D2-type with respect to Ah-locus (Nos. 2, 15, 19, 21, 22, 24, and 31), which are Ah-nonresponsive, no cleft palates occurred. One strain (No. 2) had a few (17%) fetuses with hydronephrosis. The frequency of fetal deaths and resorptions were relatively low, but slightly higher among B6-strains than D2-strains. The results indicate an association between the genes producing malformations by TCDBF and the Ah-locus.

Nerve growth factor in three neurologically deficient mouse mutants.

The activity of nerve growth factor (NGF) in the salivary glands and in the sciatic nerve was compared between normal mice and mice affected by either of three neurological mutations by the use of a biological assay. No evidence was obtained for defects in amount or activity of NGF associated with the sprawling or splotch mutations. A reduction in the NGF content was found in salivary glands and sciatic nerve in homozygous dystonia musculorum mice. It is pointed out that the low amounts of NGF in dtJ/dtJ mice is likely to be a consequence of the general disturbances in development seen in this mutant rather than the specific cause for the neurological disorder.

Pigment mutations in the mouse which also affect lysosomal functions lead to suppressed natural killer cell activity.

The impact of five pigment mutations in the mouse on natural killer (NK) activity was examined in inbred strains congenic for the respective mutation. Whereas the nature of pigmentation disorder was similar in the five mutant strains (beige, pallid, reduced pigmentation, pale ear, and sepia), all mutations except sepia also led to a significant change in lysosomal enzyme activities in the kidney. A significant reduction in NK activity was observed in the four strains with lysosomal impact, whereas homozygous sepia mice displayed normal NK activity. The pigment mutations analysed are located on different chromosomes and fail to cross-interact negatively with each other in the heterozygous mice. This would indicate that pigment mutations with a parallel impact on lysosomal enzyme activities probably always result in a reduction in natural killer cell activity.

A gene affecting liver activities of three glycosidases in the house mouse.

A gene locus is described controlling liver activities in the house mouse of three glycosidases, i.e., beta-galactosidase, beta-glucuronidase, and N-acetyl-beta-hexosaminidase. An allele conferring low activity is present in the inbred strain LIS/A, and an allele for high activity is present in A/BrAf mice. The three enzyme activities are correlated with each other. The possible linkage between this gene and the Bgs locus on chromosome 9 is discussed.

Evolutionary conservation of large chromosomal segments reflected in mammalian gene maps.

Conservation of genetic linkage over long periods of time is exemplifted. Comparisons are made between chromosomal regions in different species as well as within two species, man and the house mouse. Homologous regions are defined and the phenomenon of differential silencing of genes is described. The importance of conservation of particular sequences of genes is discussed in relation to medical genetics, animal breeding, evolutionary theory and genetic regulation.