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PubMed2001

Mathematical elegance with biochemical realism: the covarion model of molecular evolution.

There is an apparent paradox in our understanding of molecular evolution. Current biochemically based models predict that evolutionary trees should not be recoverable for divergences beyond a few hundred million years. In practice, however, trees often appear to be recovered from much older times. Mathematical models, such as those assuming that sites evolve at different rates [including a Gamma distribution of rates across sites (RAS)] may in theory allow the recovery of some ancient divergences. However, such models require that each site maintain its characteristic rate over the whole evolutionary period. This assumption, however, contradicts the knowledge that tertiary structures diverge with time, invalidating the rate-constancy assumption of purely mathematical models. We report here that a hidden Markov version of the covarion model can meet both biochemical and statistical requirements for the analysis of sequence data. The model was proposed on biochemical grounds and can be implemented with only two additional parameters. The two hidden parts of this model are the proportion of sites free to vary (covarions) and the rate of interchange between fixed sites and these variable sites. Simulation results are consistent with this approach, providing a better framework for understanding anciently diverged sequences than the standard RAS models. However, a Gamma distribution of rates may approximate a covarion model and may possibly be justified on these grounds. The accurate reconstruction of older divergences from sequence data is still a major problem, and molecular evolution still requires mathematical models that also have a sound biochemical basis.

Evolution, Molecular↗
PubMed2004

A type of nucleotide motif that distinguishes tobamovirus species more efficiently than nucleotide signatures.

The complete genomic sequences of forty-eight tobamoviruses were classified and found to form at least twelve species clusters. Individual species were not conveniently defined by 'nucleotide signatures' (i.e. strings of one or more nucleotides unique to a taxon) as these were scattered sparsely throughout the genomes and were mostly single nucleotides. By contrast all the species were concisely and uniquely distinguished by short nucleotide motifs consisting of conserved genus-specific sites intercalated with variable sites that provided species-specific combinations of nucleotides (nucleotide combination motifs; NC-motifs). We describe the procedure for finding NC-motifs in a convenient and phylogenetically conserved region of the tobamovirus RNA polymerase gene, the '4404-50 motif'. NC-motifs have been found in other sets of homologous sequences, and are convenient for use in published taxonomic descriptions.

Conserved Sequence↗
PubMed2001

A permutation procedure for the haplotype method for identification of disease-predisposing variants.

Once a genetic region involved in a complex disease has been localized through linkage or association studies, we need methods to help us identify the actual disease predisposing genetic variant(s) in the region. A large number of single nucleotide polymorphic (SNP) sites may exist in this region. It is important to identify genetic variants directly involved in disease from those in linkage disequilibrium, and thus associated with, the disease predisposing variant(s). A question of great interest is to test whether a SNP, or a combination of SNPs, that influence the trait under investigation have been identified. For many complex HLA-associated diseases, patterns of amino acid site variability raise the possibility that HLA-variation association with a disease may not be due to a given allele but rather one or more variable amino acid sites occurring on several alleles. Here the question is whether an amino acid variant or a combination of amino acid variants involved in disease are identified. To address this question, this paper proposes a permutation procedure for the haplotype method, to test whether all the sites involved in the disease have been identified using the haplotypic data of patients and controls. The method is based on the theoretical result of Valdes and Thomson, that, for each haplotype combination containing all the amino acid sites involved in the disease process, the relative frequencies of amino acid variants at sites not involved in disease, but in linkage disequilibrium with the disease-predisposing sites, are expected to be the same in patients and controls. This procedure takes into account the non-independence of the sites sampled and is robust to mode of inheritance and penetrance of the disease, and can definitely specify when all the disease predisposing sites have not been identified. Application to both simulated data and real data sets on type 1 diabetes and alcoholism indicates that the proposed procedure works well in testing the important null hypothesis of whether all the predisposing sites are identified.

Alcohol Dehydrogenase↗
PubMed2000

Heat shock protein 60 sequence comparisons: duplications, lateral transfer, and mitochondrial evolution.

Heat shock proteins 60 (GroEL) are highly expressed essential proteins in eubacterial genomes and in eukaryotic organelles. These chaperone proteins have been advanced as propitious marker sequences for tracing the evolution of mitochondrial (Mt) genomes. Similarities among HSP60 sequences based on significant segment pair alignment calculations are used to deduce associations of sequences taking into account GroEL functional/structural domain differences and to relate HSP60 duplications pervasive in alpha-proteobacterial lineages to the dynamics of lateral transfer and plasmid integration. Multiple alignments with consensuses are determined for 10 natural groups. The group consensuses sharpen the similarity contrasts among individual sequences. In particular, the Mt group matches best with the classical alpha-proteobacteria and closely with Rickettsia but significantly worse with the rickettsial groups Ehrlichia and Orientia. However, across broad protein sequence comparisons, there appears to be no consistent prokaryote whose protein sequences align best with animal Mt genomes. There are plausible scenarios indicating that the nuclear-encoded HSP60 (and HSP70) sequences functioning in Mt are results of lateral transfer and are probably derived from an alpha-proteobacterium. This hypothesis relates to the plethora of duplicated HSP60 sequences among the classical alpha-proteobacteria contrasted with no duplications of HSP60 among other clades of proteobacterial genomes. Evolutionary relations are confounded by differential selection pressures, convergence, variable mutational rates, site variability, and lateral gene transfer.

Bacteria↗
PubMed2004

Evaluation and determinants of airborne bacterial concentrations in school classrooms.

A survey of 39 elementary schools was undertaken to determine indoor air concentrations of bioaerosols within a coastal, temperate climatic zone in British Columbia, Canada. This article reports the results for airborne bacteria. Determinants of exposure were grouped into environmental (outdoor temperature, relative humidity, season, weather), ventilation and comfort parameter (indoor relative humidity, temperature, indoor CO2 concentration, indoor fungal concentration), and occupancy (number of occupants, activity levels, occupancy patterns) variables. Regression modeling was used to evaluate the association between these factors and measured concentrations of indoor mesophilic bacteria. Naturally ventilated rooms (47% of the total rooms surveyed) had higher bacterial counts than mechanically ventilated rooms (geometric mean 325 vs. 166 CFU/m3, respectively, p < 0.001). In univariate analyses, bacterial counts were negatively correlated with supply and exhaust flow rates, air exchange rates, and the percentage of the day that occupants spent quietly sitting at their desks. Analysis of bacterial groups indicated various sources of the bacterial aerosol, with micrococci and staphylococci closely associated with occupancy variables, Bacillus with site and occupancy, and coryneform bacteria with site variables only. Approximately 60% of the variance in the outcome measurement of total bacteria was accountedfor by indoor CO2, lower air exchange rate, the age of the building, signs of current or old moisture stains, room volume, indoor relative humidity, and occupant activity level in a multiple linear regression model, with ventilation factors accounting for 40% of the variance.

Aerosols↗
PubMed1998

A covariotide model explains apparent phylogenetic structure of oxygenic photosynthetic lineages.

The aims of the work were (1) to develop statistical tests to identify whether substitution takes place under a covariotide model in sequences used for phylogenetic inference and (2) to determine the influence of covariotide substitution on phylogenetic trees inferred for photosynthetic and other organisms. (Covariotide and covarion models are ones in which sites that are variable in some parts of the underlying tree are invariable in others and vice versa.) Two tests were developed. The first was a contingency test, and the second was an inequality test comparing the expected number of variable sites in two groups with the observed number. Application of these tests to 16S rDNA and tufA sequences from a range of nonphotosynthetic prokaryotes and oxygenic photosynthetic prokaryotes and eukaryotes suggests the occurrence of a covariotide mechanism. The degree of support for partitioning of taxa in reconstructed trees involving these organisms was determined in the presence or absence of sites showing particular substitution patterns. This analysis showed that the support for splits between (1) photosynthetic eukaryotes and prokaryotes and (2) photosynthetic and nonphotosynthetic organisms could be accounted for by patterns arising from covariotide substitution. We show that the additional problem of compositional bias in sequence data needs to be considered in the context of patterns of covariotide/covarion substitution. We argue that while covariotide or covarion substitution may give rise to phylogenetically informative patterns in sequence data, this may not always be so.

Markov Chains↗
PubMed1992

Quantitative determination of rare mRNA species by PCR and solid-phase minisequencing.

We present a new method for quantification of mRNA, in which the limitations of the current quantitative PCR methods can be overcome. A known amount of a synthetic RNA standard differing from the mRNA to be quantified by a single nucleotide is reverse-transcribed and amplified together with the mRNA template using a biotinylated primer. The biotinylated PCR product is immobilized on a streptavidin-coated solid support and denatured. The ratio between the two amplified sequences is determined by separate "mini-sequencing" reactions, in which a detection step primer annealing immediately adjacent to the site of the variable nucleotide is elongated by a single labeled dNTP complementary to the nucleotide at the variable site. The ratio between the incorporated labels accurately determines the ratio between the two sequences in the original RNA sample. We applied this method to quantify the mRNA of human aspartylglucosaminidase (AGA) in tissues and cultured cells. AGA is a lysosomal enzyme participating in the degradation of glycoproteins. A mutation in the AGA gene abolishes the enzyme activity and leads to aspartylglucosaminuria (AGU), a recessively inherited metabolic disorder. The mRNA quantification revealed that the normal and mutant genes are expressed at similar levels in kidney, liver, and cultured fibroblast, whereas the amount of AGA mRNA in normal placenta and brain is significantly higher than that found in the corresponding samples from AGU patients. The method presented here is generally applicable for PCR-based quantification of rare mRNAs and DNA as well.

Aspartylglucosaminuria↗
PubMed2005

A comparison of peripheral and centrally collected cyclosporine a blood levels in pediatric patients undergoing stem cell transplant.

PURPOSE/OBJECTIVES: To measure differences in cyclosporine A (CSA) trough concentrations from blood collected as a peripheral sample and from a CSA-uncontaminated (naive) lumen of a double-lumen central line. DESIGN: Prospective, comparative study. SETTING: Pediatric university teaching hospital in metropolitan Australia. SAMPLE: 71 paired central and peripheral CSA blood samples from a convenience sample of 14 pediatric allogeneic stem cell transplant recipients receiving IV CSA as prophylaxis or treatment for graft-versus-host disease. Ages ranged from 2 months to 14 years, 5 months. METHODS: Comparing blood samples collected from a peripheral site and a CSA-naive lumen of a double-lumen central line. Data were analyzed using a paired student t test and calculation of the 95% confidence interval of the concentration ratio from different sampling sites. MAIN RESEARCH VARIABLES: Site of blood sampling and CSA trough concentrations. FINDINGS: No significant difference existed between CSA concentration in samples collected from the different sites in children receiving intermittent infusions of CSA (p = 0.13). The 95% confidence interval of the CSA concentration ratio was 0.92 1.04. CONCLUSIONS: When CSA is administered on an intermittent dosing schedule, comparable CSA trough concentrations can be determined from blood collected via the CSA-naive lumen of a double-lumen central line or at a peripheral sampling site. IMPLICATIONS FOR NURSING: Pediatric allogeneic stem cell transplant recipients who require regular CSA trough concentrations no longer will require peripheral blood samples when receiving an intermittent dosing schedule.

Adolescent↗
PubMed2004

Molecular phylogeny of the nasuta subgroup of Drosophila based on 12S rRNA, 16S rRNA and CoI mitochondrial genes, RAPD and ISSR polymorphisms.

The nasuta subgroup is a cluster of morphologically almost similar forms with a wide range of geographic distribution. During the last three decades nature of inter-relationship among the members has been investigated at different levels of organization. The phylogenetic relationships of the members of the nasuta subgroup of the immigrans species group of Drosophila was made by employing Random Amplified Polymorphic DNA (RAPD), Inter Simple Sequence Repeats-PCR (ISSR-PCR) polymorphisms, mitochondrial 12S rRNA, 16S rRNA and Cytochrome C Oxidase subunit I (CoI) gene sequences. The phylogenetic tree generated by RAPD analysis is in nearly complete congruence with the classification based on morphophenotypic characters. The 12S and 16S rRNA genes were highly conserved across the nasuta subgroup and revealed only 3 and 4 variable sites respectively, of which only one site was informative. The CoI gene, on the other hand, revealed 57 variable sites of which 25 sites were informative. All the three species of orbital sheen complex were included in a major cluster in the phylogenetic trees derived from mitochondrial gene sequence data consistent with the morphophenotypic classification. The CoI analysis placed two species of frontal sheen complex, D. n. nasuta and D. n. albomicans in two different clades and this is inconsistent with morphological classification. The molecular clock suggested that divergence between the kohkoa complex and the albomicans complex occurred approximately 2.2 MYA, indicating recent evolution of the nasuta subgroup. The higher transition bias in the mitochondrial genes reported in the present study also suggested recent evolution of the nasuta subgroup.

Animals↗
PubMed2004

Selection on Glycine beta-1,3-endoglucanase genes differentially inhibited by a Phytophthora glucanase inhibitor protein.

Plant endo-beta-1,3-glucanases (EGases) degrade the cell wall polysaccharides of attacking pathogens and release elicitors of additional plant defenses. Isozymes EGaseA and EGaseB of soybean differ in susceptibility to a glucanase inhibitor protein (GIP1) produced by Phytophthora sojae, a major soybean pathogen. EGaseA, the major elicitor-releasing isozyme, is a high-affinity ligand for GIP1, which completely inhibits it, whereas EGaseB is unaffected by GIP1. We tested for departures from neutral evolution on the basis of partial sequences of EGaseA and EGaseB from 20 widespread accessions of Glycine soja (the wild progenitor of soybean), from 4 other Glycine species, and across dicotyledonous plants. G. soja exhibited little intraspecific variation at either locus. Phylogeny-based codon evolution models detected strong evidence of positive selection on Glycine EGaseA and weaker evidence for selection on dicot EGases and Glycine EGaseB. Positively selected peptide sites were identified and located on a structural model of EGase bound to GIP1. Positively selected sites and highly variable sites were found disproportionately within 4.5 angstroms of bound GIP1. Low variation within G. soja EGases, coupled with positive selection in both Glycine and dicot lineages and the proximity of rapidly evolving sites to GIP1, suggests an arms race involving repeated adaptation to pathogen attack and inhibition.

Alleles↗
PubMed2000

Molecular phylogenetic relationships of pond frogs distributed in the Palearctic region inferred from DNA sequences of mitochondrial 12S ribosomal RNA and cytochrome b genes.

The evolutionary relationships of pond frogs distributed in the Far East and Europe were investigated by analyses of nucleotide sequences of mitochondrial 12S ribosomal RNA (12S rRNA) and cytochrome b (cyt b) genes. The nucleotide sequences of a 412-bp segment of the 12S rRNA gene and a 534-bp segment of the cyt b gene were determined by the PCR-direct sequencing method using 19 frogs belonging to six species and one subspecies distributed in the Palearctic region. Phylogenetic trees were constructed by the neighbor-joining and maximum-likelihood methods using Rana catesbeiana or Xenopus laevis as an outgroup. The 412-bp segment of the 12S rRNA gene contained 65 variable sites including gap sites, and the 534-bp segment of the cyt b gene contained 160 variable sites. The nucleotide sequence divergences of the 12S rRNA gene were 0.25-4.83% within the Far Eastern frogs, 0.25-6.22% within the European frogs, and 8.74-11.24% between the Far Eastern and the European frogs, whereas those of the cyt b gene were 3.64-14.73% within the Far Eastern frogs, 0.38-14.42% within the European frogs, and 16.53-23.58% between the Far Eastern and the European frogs. Although most nucleotide substitutions were at the third codon position of the cyt b gene and were silent mutations, 4 amino acid replacements occurred within the Far Eastern frogs, 4 within the European frogs, and 11 between the Far Eastern and the European frogs. The phylogenetic trees constructed from the nucleotide sequence divergences showed slightly different topologies for the 12S rRNA and cyt b genes. R. esculenta from Ukraine was closely related to R. lessonae from Luxembourg in both the 12S rRNA and the cyt b gene sequences.

Animals↗
PubMed1998

Evolutionary relationships among Japanese pond frogs inferred from mitochondrial DNA sequences of cytochrome b and 12S ribosomal RNA genes.

The evolutionary relationships among Japanese pond frogs (Rana nigromaculata, R.porosa porosa, and R. p. brevipoda) were investigated by analyzing nucleotide sequences of mitochondrial cytochrome b (cyt b) and 12S rRNA genes. The nucleotide sequences of 444-bp segment of the cyt b gene and 410-bp segment of 12S rRNA gene were determined by the PCR-direct sequencing method using 18 frogs from 13 populations of Japanese pond frogs, and phylogenetic trees were constructed by the neighbor-joining and maximum likelihood methods using R. catesbeiana as an outgroup. The sequenced 444-bp segment of cyt b gene provided 69 variables sites, and the sequenced 410-bp segment of 12S rRNA gene provided 21 variables sites. The numbers of nucleotide substitutions per site of the cyt b gene within ingroup were 0.0022-0.0205 at the populational level, 0.0368-0.0462 at the racial or subspecific level, and 0.1038-0.1244 at the specific level, whereas those of the 12S rRNA gene were 0-0.0074 at the populational or subspecific level, and 0.0378-0.0456 at the specific level. Most nucleotide substitutions within ingroup occurred at the third codon position of the cyt b gene and were silent mutations. High frequencies of transitions relative to transversions were shown at cyt b and 12S rRNA genes within ingroup. The phylogenetic trees constructed from the nucleotide sequences of the cyt b gene showed that after outgroup R. catesbeiana separated from ingroup frogs, ingroup Japanese pond frogs diverged into R.nigromaculata and R.porosa, then the latter diverged into R.p. porosa, R.p. brevipoda (the typical Okayama race), and the Nagoya race of R.p.porosa. The phylogenetic trees constructed from the nucleotide sequences of the 12S rRNA gene also showed distinct divergence between two species, but not any divergence within species.

Amino Acid Sequence↗
PubMed2002

Risk assessment at hazardous waste-contaminated sites with variability of population characteristics.

Risk assessment is considered to be an effective scientific tool which enables decisionmakers to manage hazardous waste-contaminated sites in a cost-effective manner while preserving public health. However, the current risk assessment framework proposed by the US Environmental Protection Agency (US EPA) has limitations in addressing the true variability of population characteristics. This study proposed a methodology that is different from the existing framework by accounting for the true variability of population characteristics. The key differences of the proposed methodology and the existing framework are the (1) use of the transient exposure concentration; (2) use of the entire population rather than a representative ideal individual; (3) use of age- and gender-based population subgroups to represent population characteristics; (4) use of a population growth model to represent growth dynamics; and (5) presentation of risk through a risk profile with risk summarized through a single indicator, potential cancer incidences (PCI). The proposed methodology was applied in a ground water contamination scenario due to benzene to determine its applicability. The results of the study showed that age-based variability of population characteristics is important in predicting the population risk while gender played a small role. The existing US EPA methodology and its variation using age-independent variability of population characteristics overestimate the risk given by PCI substantially, and therefore, the decisions can lead to costly cleanup goals. Population risk is not a single value but a distribution due to the contribution from ditferent individuals of the exposed population. Hence, the decision criterion proposed in this study, PCI, is found to be a useful indicator to describe population carcinogenic risk to the society under a variety of conditions and scenarios.

Demography↗
PubMed2002

Site-specific variability in trabecular bone dosimetry: considerations of energy loss to cortical bone.

With continual advances in radionuclide therapies, increasing emphasis is being placed on improving the patient specificity of dose estimates to marrow tissues. While much work has been focused on determining patient-specific assessments of radionuclide uptake in the skeleton, few studies have been initiated to explore the individual variability of absorbed fraction data for electron and beta-particle sources in various skeletal sites. The most recent values of radionuclide S values used in clinical medicine continue to utilize a formalism in which electrons are transported under a trabecular bone geometry of infinite extent. No provisions are thus made for the fraction of energy lost to the cortical bone cortex of the skeletal site and its surrounding tissues. In the present study, NMR microscopy was performed on trabecular bone samples taken from the femoral head and humeral proximal epiphysis of three subjects: a 51-year male, an 82-year female, and an 86-year female. Following image segmentation and coupling to EGS4, electrons were transported within macrostructural models of the various skeletal sites that explicitly include the spatial extent of the spongiosa, as well as the thickness of the surrounding cortical bone. These energy-dependent profiles of absorbed fractions to marrow tissues were then compared to transport simulations made within an infinite region of spongiosa. Ratios of mean absorbed fraction, as weighted by the beta energy spectra, under both transport methodologies were then assembled for the radionuclides 32P and 90Y. These ratios indicate that corrections to existing radionuclide S values for 32P can vary by as much as 5% for the male, 6% for the 82-year female, and 8% for the 86-year female. For the higher-energy beta spectrum of 90Y, these same corrections can reach 8%, 10%, and 11%, respectively.

Aged↗
PubMed2006

Human mtDNA site-specific variability values can act as haplogroup markers.

Sequencing of entire human mtDNA genomes has become rapid and efficient, leading to the production of a great number of complete mtDNA sequences from a wide range of human populations. We introduce here a new statistical approach for classifying mtDNA nucleotide sites, simply by comparing the mean simple deviation (MSD) of their specific variability values estimated on continent-specific dataset sequences, without the need for any reference sequence. Excellent correspondence was observed between sites with the highest MSD values and those marking known mtDNA haplogroups. This in turn supports the classification of 81 sites (23 in Africa, eight in Asia, eight in Europe, 34 in Oceania, and eight in America) as novel markers of 47 mtDNA haplogroups not yet identified by phylogeographic studies. Not only does this approach allow refinement of mtDNA phylogeny, an essential requirement also for mitochondrial disease studies, but may greatly facilitate the discrimination of candidate disease-causing mutations from haplogroup-specific polymorphisms in mtDNA sequences of patients affected by mitochondrial disorders.

DNA Mutational Analysis↗
PubMed1996

Phylogeny and systematics of 18 Colletotrichum species based on ribosomal DNA spacer sequences.

The potential use of the ribosomal DNA internal transcribed spacer (ITS) sequences in understanding the phylogeny and systematics of Colletotrichum species has been evaluated. Sequence data from a limited number of isolates revealed that in Colletotrichum species the ITS 1 region (50.3% variable sites) shows a greater degree of intra- and inter-specific divergence than ITS 2 (12.4% variable sites). Nucleotide sequences of the ITS 1 region from 93 isolates representing 18 Colletotrichum species were determined. Data for 71 of these isolates where molecular and morphological identities concurred were used for phylogenetic analysis. The size of the ITS 1 region varied from 159 to 185 base pairs. Maximum intraspecific divergence was recorded with C. acutatum (5.8%), and C. capsici showed the greatest level of interspecific divergence (8.9-23.3%). Parsimony and distance analyses gave similar tree topologies. The bootstrapped consensus parsimony tree divided the 18 Colletotrichum species into six phylogenetic groups, designated 1-6. These groups, however, are not congruent with species clusterings based on spore shape. For example, the straight cylindrical spored species were represented both in groups 1 and 6; group 6 also included the falcate fusiform spored species C. capsici. The molecular evidence suggests refinement of the species concepts of some of the taxa examined. In group 6, divergence between C. gloeosporioides and C. fuscum (0.6-3.0%) or C. kahawae (0.6-3.0%) or C. fragariae (0.6-4.2%) overlap the divergence (3.6%) within C. gloeosporioides. It is suggested that C. fuscum as well as C. kahawae and C. fragariae fall within the group species C. gloeosporioides. ITS 1 data enabled clear distinction (7.1%) of Colletotrichum isolates from maize and sorghum into C. graminicola and C. sublineolum, respectively (group 2). Species such as C. acutatum, C. coccodes, C. dematium, and C. trichellum can be clearly distinguished based on ITS 1 sequence divergence, but C. destructivum cannot be confidently separated (98% homology) from C. linicola. Colletotrichum dematium f. truncatum is distinct (12.9%) from C. dematium and should probably be called C. truncatum.

Base Sequence↗
PubMed2001

Assessment of the nucleotide sequence variability in the bovine T-cell receptor alpha delta joining gene region.

The sequence of 2,193 nucleotides from the bovine T-cell receptor alpha/delta joining gene region (TCRADJ) was determined and compared with the corresponding human and murine sequences. The identity was 75.3% for the comparison of the Bos taurus vs. the Homo sapiens sequence and 63.8% for the Bos taurus vs. the Mus musculus sequence. This comparison permitted the identification of the putatively functional elements within the bovine sequence. Direct sequencing of 2,110 nucleotides in nine animals revealed 12 variable sites. Estimates, based on direct sequencing in three Holstein Friesian animals, for the two measures of sequence variability, nucleotide polymorphism (u) and nucleotide diversity (p), were 0.00050 (60.00036) and 0.00077 (60.00056), respectively. The test statistic, Tajima's D, for the comparison of the two measures indicates that the difference between u and p is close to significance (P < 0.05), suggesting the possibility of selective forces acting on the studied genomic region. Allelic variation at 5 of the 12 variable sites was analysed in 359 animals (48 Anatolian Black, 56 Braunvieh, 115 Fleckvieh, 47 Holstein Friesian, 50 Simmental and 43 Pinzgauer) using the oligonucleotide ligation assay (OLA) in combination with the enzyme linked immunoabsorbant assay (ELISA). Nine unambiguous haplotypes could be derived based on animals with a maximum of one heterozygous site. Four to seven haplotypes were present in the different breeds. When taking into account the frequencies of the haplotypes in the different breeds, especially in Anatolian Black, an ancestral cattle population, we could establish the likely phylogenetic relationships of the haplotypes. Such haplotype trees are the basis for cladistic candidate gene analysis. Our study demonstrates that the systematic search of single nucleotide polymorphisms (SNPs) is useful for analysing all aspects of variability of a given genomic region.

Alleles↗
PubMed2005

Controlling N-linked glycan site occupancy.

N-linked glycosylation, a common co-translational modification in eukaryotic cells, involves the transfer of a lipid-linked oligosaccharide onto asparagine residues in a tripeptide sequon on a nascent protein in the lumen of the endoplasmic reticulum. The attachment of an oligosaccharide unit to the polypeptide at the site of occupancy can enhance solubility, improve folding, facilitate secretion, modulate antigenicity, and increase in vivo half-life of the glycoprotein. A number of proteins exhibit variable site occupancy. The efficiency of protein N-glycosylation is dependent on the kinetics of the individual steps in the biosynthesis of the dolichol-linked oligosaccharide and the transfer of the oligosaccharide from the lipid donor substrate to the nascent polypeptide. In this review, we will discuss the role of N-linked glycan site occupancy and give an overview of the possible limitations associated with variable site occupancy. The characterization of the dolichol pyrophosphate biosynthetic pathway and the recent identification of potential rate limiting enzymes in yeast and mammalian cells has made it possible to investigate their role in site occupancy. Genetic and biochemical characterization of oligosaccharide transferase (OST) complex in yeast and mammalian cells have demonstrated the importance of specific OST subunits in protein N-glycosylation. In addition, insights into the location and residues in and around the acceptor tripeptide sequon suggest an influence on N-glycan site occupancy. Insights from these characterizations are being used to elucidate methodologies to control N-glycosylation site heterogeneity.

Animals↗