Genetic Variation in Amount of Salivary Amylase in the Bank Vole, CLETHRIONOMYS GLAREOLA

Several investigated bank vole populations are polymorphis for the number of salivary amylase loci, and individual chromosomes may carry one, two or three linked amylase structural genes. In the present study, we have used bank vole stocks homozygous for different chromosomes to investigate the relationship between amylase production and gene number. By measuring the amylase activity in parotid glands and the percentage of amylase protein in saliva, we have been able to demonstrate that the amount of salivary amylase is directly proportional to the proposed gene number. The paper also describes the allele, AmySu, which codes for a heat-labile salivary amylase. The relative amounts of the heat-labile isozyme have been determined in different heterozygotes containing this allele, and these results also support the multiple locus model. Finally, a stock devoid of salivary amylase activity was established. Animals from this strain have, however, a protein in the parotid glands and in saliva that is very similar to amylase in molecular weight, amino acid composition and in its binding to glycogen and cyclohepta-amylose. In genetic crosses, the protein segregates as an amylase allele. Therefore, this protein, encoded by the functionally null allele AmyN, may represent an incorrectly processed amylase precursor.     

MHC and Preferences for Male Odour in the Bank Vole

Highly polymorphic major histocompatibility complex (MHC) genes are thought to play a central role in the choice of genetically compatible sexual partners in some vertebrates, although the evidence is variable across species. Here, we investigate the association between similarity in the MHC region and sexual preferences in the bank vole Myodes ( =Clethrionomys ) glareolus (Arvicollinae) in a laboratory setting. Females in post-partum oestrus were given the choice between the scents of two males in a Y-maze. Both males were unrelated to the female, but differed in their MHC similarity to the female. We found that females spent more time near the scent of MHC dissimilar males than those, with whom they shared MHC alleles. This suggests that bank voles use MHC-related cues to choose compatible mates.     

Interstrain Variation in Amylase Gene Copy Number and mRNA Abundance in Three Mouse Tissues

Amylase expression in strain YBR differs in several respects from the standard mouse phenotype. The synthesis of salivary amylase is elevated twofold in YBR mice and the synthesis of pancreatic amylase is reduced to one-half the normal rate. We have compared the concentrations of amylase mRNA in the parotid, liver and pancreas of YBR mice with those in strains A/J and C3H. We observed differences in amylase mRNA abundance which can account for the levels of amylase protein synthesis in the parotid and pancreas of these strains. Unexpectedly, the concentration of amylase mRNA in the liver of YBR mice was also higher than in the other strains. Since liver amylase is transcribed from the same gene as parotid amylase, duplication of the Amy-1 locus could account for the elevated mRNA concentration in both tissues. Quantitative analysis of genomic DNA by Southern blotting provided direct evidence for duplication of Amy-1 in strain YBR.     

Problems in Estimating the Minimum Number of Genes Contributing to Quantitative Variation

This note emphasizes the problems inherent in gene counting methods like the Castle-Wright index and questions their utility.     

Genes for the SMC Family Proteins in a Common Vole Microtus arvalis

Genes for four subfamilies of SMC (structural maintenance of chromosomes) proteins have been isolated from the genome of a common vole Microtus arvalis. The high degree of homology between representatives of each SMC protein subfamily of different classes of organisms has been demonstrated. The full-sized copy of a mammalian gene encoding SMC4 protein has been isolated and analyzed for the first time. The SMC proteins enter into the composition of complexes responsible for cohesion of sister chromatids, formation of mitotic chromosomes, recombination, DNA repair, and regulation of gene expression. We discuss the possible participation of the SMC proteins in inactivation of the X chromosome in mammalian females. Common voles of genus Microtusgroup arvalis serve a unique model for the study of the inactivation process.     

Amylase Variation in the Salt Marsh Amphipod, GAMMARUS PALUSTRIS

There are two common alleles at the Amylase-2 locus in populations of Gammarus palustris, the salt marsh amphipod. Intensive sampling of individuals from two localities at Jamaica Bay revealed a consistent pattern of heterozygote deficiency.—Five possible sources of heterozygote deficiency were examined in this study. Four of them—selection against heterozygotes, null alleles at the locus, assortative mating for amylase genotype and inbreeding—are inconsistent with the evidence and are rejected. The fifth possibility, Wahlund effects due to genetic differentiation of the population, is tentatively accepted. Although there is no direct evidence for differentiation within this population, separate populations along the Eastern seaboard are highly differentiated in a nonclinal pattern. Furthermore, the Wahlund hypothesis is consistent with observations on differences in degree of deficiency exhibited among collections at Jamaica Bay.—Animals from this population exhibit feeding preferences correlated with genotype. Given the choice of two green algae, Enteromorpha or Ulva, the frequency of the slow allele among individuals choosing Enteromorpha was higher than among those choosing Ulva. This suggests that the animals assort themselves in the field into subpopulations with different allelic frequencies. This assortment could contribute to the maintenance of the polymorphism and to the observed heterozygote deficiency. We hypothesize that genotype influences behavior in this system through the action of enzyme on substrate, which determines the nature of the oligosaccharide pool liberated early in amylolysis.     

Isolation of a Novel Campylobacter jejuni Clone Associated with the Bank Vole,Myodes glareolus

Campylobacter jejuni can be isolated from different animal hosts. Various studies have used multilocus sequence typing to look for associations between particular clones of C. jejuni and specific hosts. Here, we describe the isolation of a novel clone (sequence type 3704 [ST-3704]) of C. jejuni associated with the bank vole (Myodes glareolus).Campylobacter jejuni is one of the most common causes of gastroenteritis in humans, with food (primarily chicken) believed to be the main vehicle for infection (8). Although high prevalences are found in livestock, C. jejuni has also been isolated from wildlife, including wild birds and wild mammals, and from the farm environment (1, 2, 4, 7, 11, 13). Multilocus sequence typing (MLST) has been used to study the distribution of specific clones among isolates from different hosts and the environment (1, 2, 3, 7, 10, 15, 16, 17). Such molecular epidemiological studies have provided evidence for some host-associated genotypes. For example, some MLST clonal complexes are associated with cattle (sequence type 61 [ST-61] and ST-42 complexes), whereas others are associated with wildlife, such as rabbits and wild birds, and environmental samples (ST-45, ST-177, ST-677, ST-682, and ST-952 complexes) (2, 4, 13). In addition, previously unreported sequence types have been identified in wildlife and environmental samples (4). In this study, we describe the identification of a novel strain of C. jejuni that represents a new sequence type and that is restricted primarily to one wildlife host, namely, bank voles (Myodes glareolus), from which it was isolated over a relatively wide geographic area and time period.We undertook longitudinal studies of feces collected from the sympatric wild rodents bank voles and wood mice (Apodemus sylvaticus) in a private West Cheshire (United Kingdom) woodland habitat. Feces collected from both species were analyzed for the presence of Campylobacter spp. during two sampling periods (May to July 2001 and January 2003). In 2004 (summer/autumn) and 2005 (winter/spring), cross-sectional surveys were conducted on 6 farms (5 dairy and 1 beef farm) in South Cheshire (approximately 30 km away) to investigate the role of wildlife as reservoirs of zoonotic enteric pathogens for livestock. Farms were sampled in summer/autumn on one occasion and in winter/spring on a second occasion; feces were collected from cattle, from wild mammals opportunistically, and from live-trapped rodents. For the isolation of Campylobacter spp., approximately 0.2 ml of fecal homogenate (1:1 feces in brain heart infusion broth) was added to campylobacter enrichment broth containing 5% (vol/vol) lysed horse blood (Southern Group Labs, Corby, United Kingdom), and samples were incubated at 37°C for 24 h under microaerobic conditions in a variable-atmosphere incubator (Don Whitley Scientific Ltd., Shipley, United Kingdom) before being inoculated onto campylobacter blood-free medium containing cefoperazone and amphotericin. These plates were incubated for up to 96 h at 37°C under microaerobic conditions before being examined for the presence of colonies characteristic of Campylobacter spp. All media were obtained from LabM (IDG), Bury, United Kingdom. Suspect isolates were presumptively identified as C. jejuni by Gram staining, no growth in air, and hippurate hydrolysis (6, 18) tests. For further assignment to species, cell lysates were prepared from isolates and subjected to a number of genus- and species-specific PCR assays (5, 14, 19). The whole genome sequence was obtained from bank vole strain C414 by shotgun sequencing.Of the samples obtained from woodland rodents, 23% (10/43) of bank vole samples collected from May to July 2001 and 51% (38/75) collected in January 2003 were positive for Campylobacter spp., whereas Campylobacter spp. were not recovered from any wood mouse samples (40 samples collected from May to July 2001 and 31 samples collected in January 2003). For the farm rodents (samples collected from September 2004 to April 2005), a total of 655 wood mice and 194 bank voles were sampled. In total, 18% (34/194) of bank voles were positive for Campylobacter spp., compared to 1% (6/655) of wood mice (Table ​(Table1).1). In total, 151 isolates were identified as Campylobacter spp. by using a genus-level 16S rRNA gene PCR assay (14). However, of all of these rodent isolates, only three isolates from wood mice from the farm survey could be identified as C. jejuni by species-specific PCR assays. The remaining rodent isolates from both the woodland and the farm study did not give any amplicons using species-specific PCR assays (5, 14, 19).

TABLE 1.

Number of samples from rodents captured in the woodland and farm studies, as well as from cattle in the farm study, positive for Campylobacter jejuni and specifically for ST-3704

Measurements of Salivary Alpha Amylase and Salivary Cortisol in Hominoid Primates Reveal Within-Species Consistency and Between-Species Differences

Salivary alpha amylase (sAA) is the most abundant enzyme in saliva. Studies in humans found variation in enzymatic activity of sAA across populations that could be linked to the copy number of loci for salivary amylase (AMY1), which was seen as an adaptive response to the intake of dietary starch. In addition to diet dependent variation, differences in sAA activity have been related to social stress. In a previous study, we found evidence for stress-induced variation in sAA activity in the bonobos, a hominoid primate that is closely related to humans. In this study, we explored patterns of variation in sAA activity in bonobos and three other hominoid primates, chimpanzee, gorilla, and orangutan to (a) examine if within-species differences in sAA activity found in bonobos are characteristic for hominoids and (b) assess the extent of variation in sAA activity between different species. The results revealed species-differences in sAA activity with gorillas and orangutans having higher basal sAA activity when compared to Pan. To assess the impact of stress, sAA values were related to cortisol levels measured in the same saliva samples. Gorillas and orangutans had low salivary cortisol concentrations and the highest cortisol concentration was found in samples from male bonobos, the group that also showed the highest sAA activity. Considering published information, the differences in sAA activity correspond with differences in AMY1 copy numbers and match with general features of natural diet. Studies on sAA activity have the potential to complement molecular studies and may contribute to research on feeding ecology and nutrition.     

Dramatic Number Variation of R Genes in Solanaceae Species Accounted for by a Few R Gene Subfamilies

Most disease resistance genes encode nucleotide-binding-site (NBS) and leucine-rich-repeat (LRR) domains, and the NBS-LRR encoding genes are often referred to as R genes. Using newly developed approach, 478, 485, 1,194, 1,665, 2,042 and 374 R genes were identified from the genomes of tomato Heinz1706, wild tomato LA716, potato DM1-3, pepper Zunla-1 and wild pepper Chiltepin and tobacco TN90, respectively. The majority of R genes from Solanaceae were grouped into 87 subfamilies, including 16 TIR-NBS-LRR (TNL) and 71 non-TNL subfamilies. Each subfamily was annotated manually, including identification of intron/exon structure and intron phase. Interestingly, TNL subfamilies have similar intron phase patterns, while the non-TNL subfamilies have diverse intron phase due to frequent gain of introns. Prevalent presence/absence polymorphic R gene loci were found among Solanaceae species, and an integrated map with 427 R loci was constructed. The pepper genome (2,042 in Chiltepin) has at least four times of R genes as in tomato (478 in Heinz1706). The high number of R genes in pepper genome is due to the amplification of R genes in a few subfamilies, such as the Rpi-blb2 and BS2 subfamilies. The mechanism underlying the variation of R gene number among different plant genomes is discussed.     

Modified Method for the Determination of Grain Amylase Activity by the Falling Number

The standard method for determination of amylase activity by the falling number was modified by us to study the carbohydrate–amylase complex and time course of starch hydrolysis by amylases from rye grain. The method is based on the use of potato starch as a standard substrate and aqueous extract of grain amylases as an enzyme source.     

Ultrasonic Reaction of Bank Vole Males to the Presence of Females Varying in Hormonal Activity

Rodents are known to emit ultrasounds during social interactions. Despite the evidence that ultrasonic vocalizations are emitted during sexual encounters and may play a certain role in sexual selection, only a few studies have investigated this phenomenon, mainly in laboratory rodents. We analysed the ultrasonic calls of bank vole (Myodes glareolus) males from an outbred colony in the presence of females differing in hormonal activity. Sexually experienced males were tested during interactions with naive, ovariectomized, pregnant and post‐partum oestrous females. We found that the males’ ultrasound vocalizations depend on the phase of the bank vole females’ reproductive cycle. During encounters with post‐partum oestrous females the males emitted significantly more ultrasounds, and the total duration of ultrasound vocalization was longer. The presence of post‐partum oestrous females also influenced the type of ultrasounds: the most typical constant‐frequency sounds were significantly shorter, and two additional types of ultrasounds were presented during interactions with females ready to mate: U‐shaped frequency‐modulated sounds and frequency‐modulated upsweep sounds. To our knowledge this is the first evidence that male voles of the subfamily Arvicolinae emit different types of ultrasounds in the presence of females depending on their reproductive stage. We suggest that these ultrasounds may be employed as an attractant during reproductive behaviour and that they are potentially an element of sexual selection.     

Variation in Amylase Haplotypes among Congenic Lines of the House Mouse

Pancreatic amylase in the mouse displays considerable quantitative genetic variation. Agar gel electrophoresis reveals that homozygous animals have either one form of the enzyme, type A, or two forms, type AB. Only few animals have been found that contradict this statement, namely among Mus musculus castaneous from Thailand, which has a single-banded B type. Double-banded homozygous specimens of various origins have different relative proportions of the two isoenzymes. By measuring the A:B ratios in such animals, a number of distinct haplotypes or amylase complexes, determining ratios ranging from 61% A:39%, B to 12% A:88% B, have been recognized. These complexes differ also with respect to the total amount of amylase produced. If the reference stock C3H/As is given the value 1, then other haplotypes have values ranging from 1.0 to 0.27. Nineteen amylase haplotypes have been established in congenic lines on a C3H/As background. Some of these lines contain at least four active pancreatic amylase structural genes and breeding experiments have demonstrated that the genetic elements regulating total amylase production and relative proportions of the isoenzymes are located within the amylase complex, cis-acting, and very closely linked to the structural genes.     

整合分析基因表达与拷贝数变异识别癌症的驱动基因及调控子miRNAs

目的:通过整合分析基因的表达与拷贝数变异(CNV)识别癌症的驱动基因及调控子mi RNAs。方法:通过整合基因表达与CNV数据,分别计算了乳腺癌、结肠癌、肺癌、肾癌、膀胱癌、头颈癌六种癌症中mi RNAs的调控得分,提出了一个识别驱动基因和显著调控子mi RNAs的方法。结果:本文研究发现,CNV区域上编码的基因相比于非CNV区域上编码的基因更倾向于受mi RNAs调控。但是,癌相关CNV区域上的基因相比正常CNV区域上的基因更少受mi RNAs调控。本研究识别出了EXOSC4、ZNF7、BOP1等原癌基因,以及mi R-488、mi R-27a、mi R-454等在多种癌症中都起调控作用的调控子mi RNAs。结论:本文的方法为癌症研究带来了新的启发,这些具有调控扩增基因过表达作用的mi RNAs的发现,有助于我们更进一步了解癌基因表达的复杂调控机制,进而推动癌症的诊断、治疗和预后。     

Modifications in Estimating the Number of Genes for a Quantitative Character

In estimating the minimum number of genes contributing to a quantitative character, it is suggested that the squared difference between the means of the two parents be corrected for experimental variance and that the genetic variance stemming from differences in gene frequencies of the parents be estimated by least squares utilizing information on all entries.     

Copy Number Variation of the Beta-Defensin Genes in Europeans: No Supporting Evidence for Association with Lung Function,Chronic Obstructive Pulmonary Disease or Asthma

Lung function measures are heritable, predict mortality and are relevant in diagnosis of chronic obstructive pulmonary disease (COPD). COPD and asthma are diseases of the airways with major public health impacts and each have a heritable component. Genome-wide association studies of SNPs have revealed novel genetic associations with both diseases but only account for a small proportion of the heritability. Complex copy number variation may account for some of the missing heritability. A well-characterised genomic region of complex copy number variation contains beta-defensin genes (DEFB103, DEFB104 and DEFB4), which have a role in the innate immune response. Previous studies have implicated these and related genes as being associated with asthma or COPD. We hypothesised that copy number variation of these genes may play a role in lung function in the general population and in COPD and asthma risk. We undertook copy number typing of this locus in 1149 adult and 689 children using a paralogue ratio test and investigated association with COPD, asthma and lung function. Replication of findings was assessed in a larger independent sample of COPD cases and smoking controls. We found evidence for an association of beta-defensin copy number with COPD in the adult cohort (OR = 1.4, 95%CI:1.02–1.92, P = 0.039) but this finding, and findings from a previous study, were not replicated in a larger follow-up sample(OR = 0.89, 95%CI:0.72–1.07, P = 0.217). No robust evidence of association with asthma in children was observed. We found no evidence for association between beta-defensin copy number and lung function in the general populations. Our findings suggest that previous reports of association of beta-defensin copy number with COPD should be viewed with caution. Suboptimal measurement of copy number can lead to spurious associations. Further beta-defensin copy number measurement in larger sample sizes of COPD cases and children with asthma are needed.     

Copy Number Variation of Fc Gamma Receptor Genes in HIV-Infected and HIV-Tuberculosis Co-Infected Individuals in Sub-Saharan Africa

AIDS, caused by the retrovirus HIV, remains the largest cause of morbidity in sub-Saharan Africa yet almost all genetic studies have focused on cohorts from Western countries. HIV shows high co-morbidity with tuberculosis (TB), as HIV stimulates the reactivation of latent tuberculosis (TB). Recent clinical trials suggest that an effective anti-HIV response correlates with non-neutralising antibodies. Given that Fcγ receptors are critical in mediating the non-neutralising effects of antibodies, analysis of the extensive variation at Fcγ receptor genes is important. Single nucleotide variation and copy number variation (CNV) of Fcγ receptor genes affects the expression profile, activatory/inhibitory balance, and IgG affinity of the Fcγ receptor repertoire of each individual. In this study we investigated whether CNV of FCGR2C, FCGR3A and FCGR3B as well as the HNA1 allotype of FCGR3B is associated with HIV load, response to highly-active antiretroviral therapy (HAART) and co-infection with TB. We confirmed an effect of TB-co-infection status on HIV load and response to HAART, but no conclusive effect of the genetic variants we tested. We observed a small effect, in Ethiopians, of FCGR3B copy number, where deletion was more frequent in HIV-TB co-infected patients than those infected with HIV alone.     

Copy Number Variation in the Horse Genome

We constructed a 400K WG tiling oligoarray for the horse and applied it for the discovery of copy number variations (CNVs) in 38 normal horses of 16 diverse breeds, and the Przewalski horse. Probes on the array represented 18,763 autosomal and X-linked genes, and intergenic, sub-telomeric and chrY sequences. We identified 258 CNV regions (CNVRs) across all autosomes, chrX and chrUn, but not in chrY. CNVs comprised 1.3% of the horse genome with chr12 being most enriched. American Miniature horses had the highest and American Quarter Horses the lowest number of CNVs in relation to Thoroughbred reference. The Przewalski horse was similar to native ponies and draft breeds. The majority of CNVRs involved genes, while 20% were located in intergenic regions. Similar to previous studies in horses and other mammals, molecular functions of CNV-associated genes were predominantly in sensory perception, immunity and reproduction. The findings were integrated with previous studies to generate a composite genome-wide dataset of 1476 CNVRs. Of these, 301 CNVRs were shared between studies, while 1174 were novel and require further validation. Integrated data revealed that to date, 41 out of over 400 breeds of the domestic horse have been analyzed for CNVs, of which 11 new breeds were added in this study. Finally, the composite CNV dataset was applied in a pilot study for the discovery of CNVs in 6 horses with XY disorders of sexual development. A homozygous deletion involving AKR1C gene cluster in chr29 in two affected horses was considered possibly causative because of the known role of AKR1C genes in testicular androgen synthesis and sexual development. While the findings improve and integrate the knowledge of CNVs in horses, they also show that for effective discovery of variants of biomedical importance, more breeds and individuals need to be analyzed using comparable methodological approaches.