Polymorphism and chromosomal location of the MC4R (melanocortin-4 receptor) gene in the dog and red fox

The melanocortin-4 receptor (MC4R) is expressed in the hypothalamus and regulates energy intake and body weight. In silico screening of the canine chromosome 1 sequence and a comparison with the porcine MC4R sequence by BLAST were performed. The nucleotide sequence of the whole coding region and 3'- and 5'-flanking regions of the dog (1214 bp) and red fox (1177 bp) MC4R gene was established and high conservation of the nucleotide sequences was revealed (99%). Five sets of PCR primers were designed and a search for polymorphism was performed by the SSCP technique in a group of 31 dogs representing nineteen breeds and 35 farm red foxes. Sequencing of DNA fragments, representing the identified SSCP patterns, revealed three single nucleotide polymorphisms (including a missense one) in dogs and four silent SNPs in red foxes. An average SNP frequency was approx. 1/400 bp in the dog and 1/300 bp in the red fox. We mapped the MC4R gene by FISH to the canine chromosome 1 (CFA1q1.1) and to the red fox chromosome 5 (VVU5p1.2).     

Association of MC3R gene polymorphisms with body weight in the red fox and comparative gene organization in four canids

There are five genes encoding melanocortin receptors. Among canids, the genes have mainly been studied in the dog (MC1R, MC2R and MC4R). The MC4R gene has also been analysed in the red fox. In this report, we present a study of chromosome localization, comparative sequence analysis and polymorphism of the MC3R gene in the dog, red fox, arctic fox and Chinese raccoon dog. The gene was localized by FISH to the following chromosome: 24q24‐25 in the dog, 14p16 in the red fox, 18q13 in the arctic fox and NPP4p15 in the Chinese raccoon dog. A high identity level of the MC3R gene sequences was observed among the species, ranging from 96.0% (red fox – Chinese raccoon dog) to 99.5% (red fox – arctic fox). Altogether, eight polymorphic sites were found in the red fox, six in the Chinese raccoon dog and two in the dog, while the arctic fox appeared to be monomorphic. In addition, association of several polymorphisms with body weight was analysed in red foxes (the number of genotyped animals ranged from 319 to 379). Two polymorphisms in the red fox, i.e. a silent substitution c.957A>C and c.*185C>T in the 3′‐flanking sequence, showed a significant association (P < 0.01) with body weight.     

PRINS detection of 18S rDNA in pig, red fox and Chinese raccoon dog, and centromere DNA in horse

The fluorescence in situ hybridization (FISH) technique is widely used in animal cytogenetics. Contrary to FISH procedure, primed in situ DNA synthesis (PRINS) does not require the DNA probe preparation (design, synthesis, gel purification of PCR products and labeling). The PRINS method with primers used as 'DNA probes' is both PCR-sensitive and allows for chromosomal localization of DNA sequences. Here, we show the application of PRINS reaction with one unlabeled oligonucleotide pair to identify 18S rDNA loci in three different animal species: domestic pig (Sus scrofa), red fox (Vulpes vulpes) and Chinese raccoon dog (Nyctereutes procyonoides procyonoides). We present the data of indirect labeling with the digoxigenin-PRINS using two different pairs of primers complementary to centromeric region of horse (Equus caballus) chromosomes. Our new PRINS application may be considered as a useful tool for chromosome investigation in the field of domestic and wild animal genetics and evolution.     

圈养狐貉源犬瘟热病毒地方分离株H基因的遗传变异分析

本研究于2004～2006年从人工饲养的发病狐貉中分离到6个CDV分离株,用RT-PCR方法扩增了其H蛋白基因片段,并对其进行了克隆和测序.测序结果表明,6个CDV分离株H基因阅读框全长均为1 824 bp,编码607个氨基酸,未发现碱基的插入和缺失.与Genbank中的34株CDV参考毒株的H基因序列进行比较和分析,现有的CDV毒株可以分为Asia-1、Asia-2、America-1、America-2、Europe和Arctic 6个基因型,本实验中的分离株HL为Arctic基因型,与来自意大利的194/97株、丹麦的Green株同分在一组,其余5株分离株与来自日本的HAMA、UENO、Tanu96等毒株以及来自中国的TN株、GP株(大熊猫株)分在一组同属Asia-1型,表现出一定的地理位置相关性;6个分离株均与Onderstepoor、Convac等疫苗株差异较大、关系较远,提示病毒变异可能是造成目前已免疫动物仍然发生犬瘟热流行的原因之一.     

cDNA sequence and chromosomal localization of the mouse parvalbumin gene, Pva

In the homozygous condition, the mutation adr (arrested development of righting response) of the mouse causes a myotonia and a drastic reduction of the Ca2+-binding protein parvalbumin (PV) in fast muscles. Using a rat PV probe, a mouse cDNA clone was isolated from a lambda gt11 wild-type fast-muscle library and its nucleotide sequence was determined. The protein coding and the 3' nontranslated regions of the mouse gene show extensive homology with the rat PV gene. The result of Southern blot hybridization is consistent with a single copy gene for parvalbumin. Restriction fragment length polymorphisms (RFLPs) between Mus musculus domesticus (e.g. C57BL/6) and Mus spretus (SPE) were detected with the enzymes Eco RI, Pst I, and Sst I. The restriction fragment patterns of DNA samples from 65 individual offspring of (C57BL/6 x SPE)F1 x C57BL/6 backcrosses were tested with the PV probe and matched, for linkage detection, to pre-existing patterns established with various RFLP probes on the same samples. A co-distribution of PV-RFLPs with Pvt-1 and Mlvi-2, which had been localized on chromosome 15, was detected. Thus, the structural gene for PV, designated Pva, maps to chromosome 15 of the mouse whereas the adr mutation shows no linkage with markers on this chromosome. Gene locus homology between chromosome 15 of the mouse and chromosome 22 of man (which carries the human PV gene) is discussed.     

Canine-derived cosmid probes containing microsatellites can be used in physical mapping of Arctic fox (Alopex lagopus) and Chinese raccoon dog (Nyctereutes procyonoides procyonoides) genomes

Rapid development of the canine marker genome map facilitates genome mapping of other Canidae species. In this study we present chromosomal localization of 18 canine-derived cosmid probes containing microsatellites in the arctic fox (Alopex lagopus) and Chinese raccoon dog (Nyctereutes procyonoides procyonoides) genomes by the use of fluorescence in situ hybridization (FISH). The chromosome localizations in the arctic fox are in general agreement with data obtained from comparative genome maps of the dog and the fox. However, our studies showed that the order of the loci on some chromosomes was changed during karyotype evolution. Therefore, we suggest that small intrachromosomal rearrangements took place.     

Summer food composition and food niche overlap of the raccoon dog, red fox and badger in Finland

Diets of raccoon dog, red fox and badger were studied in southern Finland from 1990 to 1996 by collecting faeces from dens and latrines during May-July of each year. The frequency of occurrence and relative volume of each food item in the faeces were calculated. The raccoon dog was the most and the red fox the least omnivorous of these carnivores, according to the diversity index. Diet composition of all these species varied among areas, indicating that they are opportunistic feeders. Mammals and birds constituted the bulk of the fox diet, while invertebrates, frogs and plants were most frequently eaten by the badger. The frequency of earthworms in badger faeces varied between 16 and 77%, according to area. Voles and shrews were important to the raccoon dog, but it also frequently consumed other food items. The food niches of the badger and the raccoon dog overlapped more than that of the red fox and other species. The red fox was more carnivorous and fed on larger prey items than the others, but the food composition of the red fox in the present study reflects the prey carried to the den for the pups; thus, the diet of adult foxes may be more similar to that of the raccoon dog. In conclusion, these 3 carnivores share many resources, suggesting that competition may occur among them: however, their diets also differ to some extent, which helps them to avoid competition. Furthermore, the badger and the raccoon dog are dormant during winter, when food is scarcest, which may be the reason why all these species can coexist in rather unproductive boreal forests.     

A polymorphism study of the human Agouti gene and its association with MC1R

To determine whether the Agouti Signalling Protein (ASP) gene is associated with skin and hair coloration in humans, the complete coding region of ASP was screened for polymorphisms. Analysis of ASP in Caucasian, African-American, Spanish Basque, Hispanic, Apache and Australian Aboriginal populations revealed no amino acid substitutions. A single polymorphism in the 3' untranslated region occurred at a frequency of 0.2 in African-Americans. Variants of the Melanocortin 1 Receptor (MC1R) gene have been found to be associated with red hair and fair skin in humans. Red hair individuals are usually compound heterozygotes or homozygous for one of a number of MC1R polymorphisms associated with red hair. Some individuals however are heterozygous for only one of these polymorphisms and dizygotic twins can be concordant for MC1R variants but discordant for hair colour. A recent study has also identified rare redheads carrying no MC1R variants indicating that polymorphisms of the human MC1R gene are required but not sufficient for the red hair phenotype. To address the question of whether ASP also contributes to the red hair phenotype, individuals previously identified as having unexpected MC1R genotypes were screened for polymorphisms at the ASP locus. No polymorphisms were found in any of these individuals. Results indicate that the human ASP gene is unlikely to function in normal human pigmentation in the same way as MC1R.     

北极狐GHR基因cDNA的克隆及序列分析

本文根据狗(AF133835)的GHR基因cDNA编码全序列设计了三对引物,利用RT-PCR方法克隆出北极狐GHR基因编码区全长cDNA序列(GenBank accession No.EU304325)。结果表明,北极狐GHR的ORF为1917bp,编码638个氨基酸的前体蛋白,由18个氨基酸的信号肽和620个氨基酸的成熟肽组成。通过同源性比较发现北极狐与狗的同源性最高,达到98%。另外,利用邻接法(NJ法)构建的分子系统进化树聚类结果表明,北极狐与狗先聚为一类,该聚类结果与传统的物种进化关系基本一致。另外,通过氨基酸对位序列比较发现,北极狐GHR在氨基酸序列上存在明显的特异性,如45和451位分别为A和E,而其它物种均分别为T(大鼠为K)和A(牛羊为V,鼠为T)。     

The human glucagon receptor encoding gene: structure, cDNA sequence and chromosomal localization

Characterization of the human glucagon-receptor-encoding gene (GGR) should provide a greater understanding of blood glucose regulation and may reveal a genetic basis for the pathogenesis of diabetes. A cDNA encoding a complete functional human glucagon receptor (GGR) was isolated from a liver cDNA library by a combination of polymerase chain reaction and colony hybridization. The cDNA encodes a receptor protein with 80% identity to rat GGR that binds [¹²⁵I] glucagon and transduces a signal leading to increases in the concentration of intracellular cyclic adenosine 3′,5′-monophosphate. Southern blot analysis of human DNA reveals a hybridization pattern consistent with a single GGR locus. In situ hybridization to metaphase chromosome preparations maps the GGR locus to chromosome 17q25. Analysis of the genomic sequence shows that the coding region spans over 5.5 kb and is interrupted by 12 introns.     

Analysis of the unassembled part of the dog genome sequence: chromosomal localization of 115 genes inferred from multispecies comparative genomics

The identification of dog genes and their accurate localization to chromosomes remain a major challenge in the postgenomics era. The 132 annotated canine genes with human orthologs remaining in the unassembled part (chrUnknown) of the dog sequence assembly (CanFam1) are of limited use for candidate gene approaches or comparative mapping studies. We used a two-step comparative analysis to infer a canine chromosomal interval for localization of the chrUn genes. We first constructed a human-dog synteny map, using 14,456 gene-based comparative anchors. We then mapped the 132 chrUn genes onto the reference (human) synteny map and identified the corresponding, orthologous segment on the canine map, based on conserved gene order. Our results show that 110 chrUn genes could be localized to short intervals on 18 dog chromosomes, whereas 22 genes remained assigned to 2 possible intervals. We extended this comparative analysis to multiple species, using the chimpanzee, mouse, and rat genome sequences. This made it possible to narrow down the intervals concerned and to increase the number of canine chrUn genes with an inferred chromosome location to 115. This study demonstrates that dog chromosomal intervals for chrUn genes can be rapidly inferred, using a reference species, and indicates that comparative strategies based on larger numbers of species may be even more effective.     

Nucleotide sequence, chromosomal localization and developmental expression of the mouse int-1-related gene

cDNA clones encoding the murine int-1-related protein (m-irp) were isolated from an 8.5-day mouse embryo library. m-irp and its human counterpart, h-irp, share extensive nucleotide homology in coding (92%) and 3' untranslated (69%) regions. At the amino acid level, m-irp and h-irp share 97% of amino acids including all 24 cysteine residues, which are highly conserved among members of the int-1 family. However, in contrast to h-irp and int-1, the predicted m-irp protein sequence did not contain a signal peptide sequence. Analysis of polymerase chain reaction, amplified cDNA, and genomic sequences strongly suggests that a single-base substitution has created a new 5' splice site 17 bp 5' of a highly conserved splice site. Splicing at this new site generates a mRNA-encoding an amino-terminal truncated protein. Splicing at the conserved splice site generates a mRNA species encoding a protein with a signal peptide sequence similar to h-irp. Close linkage between m-irp and the met oncogene maps m-irp sequences to proximal mouse chromosome 6. Adult and fetal expression of m-irp was examined by RNA blot analysis. Adult expression of m-irp is restricted to lungs and heart, and fetal expression, to placental tissue and to all stages of fetal development examined. In situ hybridization localized early fetal m-irp expression to the pericardium of the heart, to the umbilicus and associated allantoic mesoderm, and to the ventral lateral mesenchyme tissue surrounding the umbilical vein in the fetus. These results suggest a role for m-irp in the development of fetal allantoic communication.     

Entire sequence of a mouse chromosomal segment containing the gene Rhced and a comparative analysis of the homologous human sequence

The mouse genomic sequence of the region containing the gene Rhced, the orthologue to the human gene RH30, was determined to elucidate the structure of Rhced and its flanking regions and to compare these with the corresponding human genomic region. Two genes, Smp1 and AK003528 (an orthologue of FLJ10747), flank Rhced. Neither sequences homologous to the characteristic nucleotide elements flanking the RHD gene in humans (rhesus boxes) nor an additional Rh gene were found within the mouse region sequenced. This result and that of a previous report demonstrate that this chromosomal region of the mouse comprises five genes (FLJ10747-RHCE-SMP1-NPD014-P29) that exhibit syntenic homology with the corresponding human region, which suggests that the RHD gene and rhesus boxes were inserted later. Evaluations of tissue distribution and subcellular localization of these genes indicate that the SMP1 orthologue has a ubiquitous tissue distribution and cytoplasmic localization, whereas AK003528 is expressed slightly higher in testis with a strong subcellular localization in the nucleus. Despite the steady improvements in the draft sequence of the human genome, this study demonstrates the continuing benefits of comparative genetic analyses in increasing our understanding of human genomic structure.