Banding Homologies Research Articles

EVALUATION OF GENETIC DIVERSITY OF SESAME BY THE STUDY OF SEED STORAGE PROTEIN THROUGH SDS-PAGE

The sesame seed protein was electrophoretically separated through sodium dodecylesulphate polyacrylamide gel electrophoresis (SDS-PAGE). Electrophoretic banding homology among the genotypes was established not only through the banding pattern but also through estimation of dissimilarity percentage in protein profile. The advantage of SDS–PAGE is that the proteins are separated by their molecular weight and it has become one of the most widely used technique for storage protein separation. Freshly harvested seeds of 21 advance lines and their 8 parental lines were taken for the experiment of our study. The genetic diversity and the relationships among the 29 genotypes were evaluated using sodium dodecylesulphate polyacrylamide gel electrophoresis (SDS –PAGE) which shows difference in number of bands, band width and intensity for different genotypes. It was possible to identify as many as 21 different bands and these can be easily visualized. Dendogram of 21 advance lines and their 8 parents based on SDS – PAGE data using dissimilarity percentage was presented here. Mainly two clusters obtained from the dendrogram for all the 29 genotypes where cluster A forms 3 and cluster B forms 6 sub- clusters. Clustering pattern, size and constituents on the basis of electrophoretic banding pattern was independent of the same through the D² analysis on the basis of biochemical parameters and presence of distinct phylogenetic relationship could be recognized among the genotypes. The dissimilarity % in protein profile indicated that the B- 9 vs. adv. line 5, B-9 vs. adv. line 8, R – 9 vs. adv. line 8, adv. Line 14 vs. adv.18, HT-1 vs. adv. 11 would be useful in identifying parents for heterosis breeding.

CYTOLOGICAL STUDIES ON SOME ENDEMIC TERRESTRIAL ORCHIDS

The sesame seed protein was electrophoretically separated through sodium dodecylesulphate polyacrylamide gel electrophoresis (SDS-PAGE). Electrophoretic banding homology among the genotypes was established not only through the banding pattern but also through estimation of dissimilarity percentage in protein profile. The advantage of SDS–PAGE is that the proteins are separated by their molecular weight and it has become one of the most widely used technique for storage protein separation. Freshly harvested seeds of 21 advance lines and their 8 parental lines were taken for the experiment of our study. The genetic diversity and the relationships among the 29 genotypes were evaluated using sodium dodecylesulphate polyacrylamide gel electrophoresis (SDS –PAGE) which shows difference in number of bands, band width and intensity for different genotypes. It was possible to identify as many as 21 different bands and these can be easily visualized. Dendogram of 21 advance lines and their 8 parents based on SDS – PAGE data using dissimilarity percentage was presented here. Mainly two clusters obtained from the dendrogram for all the 29 genotypes where cluster A forms 3 and cluster B forms 6 sub- clusters. Clustering pattern, size and constituents on the basis of electrophoretic banding pattern was independent of the same through the D² analysis on the basis of biochemical parameters and presence of distinct phylogenetic relationship could be recognized among the genotypes. The dissimilarity % in protein profile indicated that the B- 9 vs. adv. line 5, B-9 vs. adv. line 8, R – 9 vs. adv. line 8, adv. Line 14 vs. adv.18, HT-1 vs. adv. 11 would be useful in identifying parents for heterosis breeding.

Functional Elucidation of Nemopilema nomurai and Cyanea nozakii Nematocyst Venoms’ Lytic Activity Using Mass Spectrometry and Zymography

Background: Medusozoans utilize explosively discharging penetrant nematocysts to inject venom into prey. These venoms are composed of highly complex proteins and peptides with extensive bioactivities, as observed in vitro. Diverse enzymatic toxins have been putatively identified in the venom of jellyfish, Nemopilema nomurai and Cyanea nozakii, through examination of their proteomes and transcriptomes. However, functional examination of putative enzymatic components identified in proteomic approaches to elucidate potential bioactivities is critically needed. Methods: In this study, enzymatic toxins were functionally identified using a combined approach consisting of in gel zymography and liquid chromatography tandem mass spectrometry (LC-MS/MS). The potential roles of metalloproteinases and lipases in hemolytic activity were explored using specific inhibitors. Results: Zymography indicated that nematocyst venom possessed protease-, lipase- and hyaluronidase-class activities. Further, proteomic approaches using LC-MS/MS indicated sequence homology of proteolytic bands observed in zymography to extant zinc metalloproteinase-disintegrins and astacin metalloproteinases. Moreover, pre-incubation of the metalloproteinase inhibitor batimastat with N. nomurai nematocyst venom resulted in an approximate 62% reduction of hemolysis compared to venom exposed sheep erythrocytes, suggesting that metalloproteinases contribute to hemolytic activity. Additionally, species within the molecular mass range of 14–18 kDa exhibited both egg yolk and erythrocyte lytic activities in gel overlay assays. Conclusion: For the first time, our findings demonstrate the contribution of jellyfish venom metalloproteinase and suggest the involvement of lipase species to hemolytic activity. Investigations of this relationship will facilitate a better understanding of the constituents and toxicity of jellyfish venom.

A Proteomic Style Approach To Characterize a Grass Mix Product Reveals Potential Immunotherapeutic Benefit

Background Grass allergy immunotherapies often consist of a mix of different grass extracts, each containing several proteins of different physiochemical properties; however, the subtle contributions of each protein are difficult to elucidate. This study aimed to identify and characterize the group 1 and 5 allergens in a 13 grass extract and to standardize the extraction method.Methods The grass pollens were extracted in isolation and pooled and also in combination and analyzed using a variety of techniques including enzyme-linked immunosorbent assay, liquid chromatography-mass spectrometry, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis.Results Gold-staining and IgE immunoblotting revealed a high degree of homology of protein bands between the 13 species and the presence of a densely stained doublet at 25-35 kD along with protein bands at approximately 12.5, 17, and 50 kD. The doublet from each grass species demonstrated a high level of group 1 and 5 interspecies homology. However, there were a number of bands unique to specific grasses consistent with evolutionary change and indicative that a grass mix immunotherapeutic could be considered broad spectrum.Conclusions Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and IgE immunoblotting showed all 13 grasses share a high degree of homology, particularly in terms of group 1 and 5 allergens. IgE and IgG enzyme-linked immunosorbent assay potencies were shown to be independent of extraction method.

Cytogenetic characterization of the moray eel Gymnothorax tile and chromosomal banding comparison in Muraenidae (Anguilliformes)

The karyotype of the teleostean Gymnothorax tile was analysed by replication banding in order to clearly identify the homologous chromosomes. The RBG pattern was then compared to those of the other Muraenidae studied (G. unicolor and Muraena helena) and banding homologies of nine chromosome pairs were pointed out. A single nucleolar organizer region (NOR) was localized by FISH, silver- and CMA3-staining on the short arm of the pair 12. Telomeric (TTAGGG) repeats were terminally localized by FISH on all the chromosome pairs. The genome size and the AT-DNA content were evaluated by flow cytometry. Available cytogenetic data on the Muraenidae were then compared and discussed.

Evaluating the Relationship between Evolutionary Divergence and Phylogenetic Accuracy in AFLP Data Sets

Using in silico amplified fragment length polymorphism (AFLP) fingerprints, we explore the relationship between sequence similarity and phylogeny accuracy to test when, in terms of genetic divergence, the quality of AFLP data becomes too low to be informative for a reliable phylogenetic reconstruction. We generated DNA sequences with known phylogenies using balanced and unbalanced trees with recent, uniform and ancient radiations, and average branch lengths (from the most internal node to the tip) ranging from 0.02 to 0.4 substitutions per site. The resulting sequences were used to emulate the AFLP procedure. Trees were estimated by maximum parsimony (MP), neighbor-joining (NJ), and minimum evolution (ME) methods from both DNA sequences and virtual AFLP fingerprints. The estimated trees were compared with the reference trees using a score that measures overall differences in both topology and relative branch length. As expected, the accuracy of AFLP-based phylogenies decreased dramatically in the more divergent data sets. Above a divergence of approximately 0.05, AFLP-based phylogenies were largely inaccurate irrespective of the distinct topology, radiation model, or phylogenetic method used. This value represents an upper bound of expected tree accuracy for data sets with a simple divergence history; AFLP data sets with a similar divergence but with unbalanced topologies and short ancestral branches produced much less accurate trees. The lack of homology of AFLP bands quickly increases with divergence and reaches its maximum value (100%) at a divergence of only 0.4. Low guanine-cytosine (GC) contents increase the number of nonhomologous bands in AFLP data sets and lead to less reliable trees. However, the effect of the lack of band homology on tree accuracy is surprisingly small relative to the negative impact due to the low information content of AFLP characters. Tree-building methods based on genetic distance displayed similar trends and outperformed parsimony at low but not at high divergences. However, the impact of using alternative phylogenetic methods on tree accuracy was generally small relative to the uncertainty arising from factors such as divergence, nonhomology of bands, or the low information content of AFLP characters. Nevertheless, our data suggest that under certain circumstances, AFLPs may be suitable to reconstruct deeper phylogenies than usually accepted.

Comparative chromosome studies in Pinnipedia.

Cells in short-term culture from 12 species of pinnipeds were submitted to chromosome analysis, including, in 11 of them, chromosome measurements and establishment of idiograms. Analysis of variance was carried out on the measurements of 9 phocids. Comparisons between the different karyotypes were made on the basis of conventional chromosome methods as well as autoradiography, G- and C-band techniques. The Pinnipedia are characterized by a pronounced karyotype uniformity. Within the Otarioidea, all otariids so far studied have 2n = 36 and closely agreeing karyotypes, the walrus has 2n = 32. In the Phocidae 2 chromosome numbers occur, 2n = 34 and 2n = 32. The 2n = 32 karyotype has evolved from the primordial 2n = 34 karyotype through fusion of two pairs. Within each of these two groups all karyotypes are virtually identical. The morphological and banding homologies between the otariid and 34 chromosome phocid karyotypes confirm the theory of monophyletic origin of the Pinnipedia.

The phylogeny of nine species of the Drosophila obscura group inferred by the banding homologies of chromosomal regions

The phylogenetic relationships among nine species of Drosophila belonging to the obscura group were investigated by establishing (according to their banding similarities) the homologous chromosome segments of element E (equivalent to chromosome O of D. subobscura). The phylogenetic relationships were based on the existence of segments in different triads of species, which could only be produced by overlapping inversions. This permitted the ordering of the species belonging to each triad. Drosophila obscura, D. ambigua and D. tristis were found to be very closely related and thus forming a cluster in which D. ambigua occupies an intermediate position between the other two species. Drosophila obscura seems to be the species more directly linked to three other separate lineages, that of D. subsilvestris, the two African species (D. microlabis and D. kitumensis), and the subobscura cluster. The species from this last cluster may be ordered as follows: D. subobscura-D. madeirensis-D. guanche. It is not clear which species of this triad is the direct link to D. obscura. These results completely agree with those produced in an independent study, where element B was considered for the same nine species. Furthermore, the present study clarifies some ambiguities concerning the phylogenetic relationships which remained obscure due to the conservative nature of chromosome B.

Impact of Amplified Fragment Length Polymorphism Size Homoplasy on the Estimation of Population Genetic Diversity and the Detection of Selective Loci

AFLP markers are becoming one of the most popular tools for genetic analysis in the fields of evolutionary genetics and ecology and conservation of genetic resources. The technique combines a high-information content and fidelity with the possibility of carrying out genomewide scans. However, a potential problem with this technique is the lack of homology of bands with the same electrophoretic mobility, what is known as fragment-size homoplasy. We carried out a theoretical analysis aimed at quantifying the impact of AFLP homoplasy on the estimation of within- and between-neutral population genetic diversity in a model of a structured finite population with migration among subpopulations. We also investigated the performance of a currently used method (DFDIST software) to detect selective loci from the comparison between genetic differentiation and heterozygosis of dominant molecular markers, as well as the impact of AFLP homoplasy on its effectiveness. The results indicate that the biases produced by homoplasy are: (1) an overestimation of the frequency of the allele determining the presence of the band, (2) an underestimation of the degree of differentiation between subpopulations, and (3) an overestimation or underestimation of the heterozygosis, depending on the allele frequency of the markers. The impact of homoplasy is quickly diminished by reducing the number of fragments analyzed per primer combination. However, substantial biases on the expected heterozygosity (up to 15-25%) may occur with approximately 50-100 fragments per primer combination. The performance of the DFDIST software to detect selective loci from dominant markers is highly dependent on the number of selective loci in the genome and their average effects, the estimate of genetic differentiation chosen to be used in the analysis, and the critical bound probability used to detect outliers. Overall, the results indicate that the software should be used with caution. AFLP homoplasy can produce a reduction of up to 15% in the power to detect selective loci.

Chromosomal localization of the glucose phosphate isomerase (GPI) gene in cattle, sheep and goat by in situ hybridization - chromosomal banding homology versus molecular conservation in Bovidae

A porcine genomic glucose phosphate isomerase (GPI) DNA probe was used for in situ hybridization with metaphase chromosomes in cattle, sheep and goat. The probe gave distinct signals on the q22----proximal part of the q24 segment of chromosome 18, 14 and 18 in cattle, sheep and goat, respectively, indicating the location of GPI gene. The three species belong to the family Bovidae and have high resemblance in chromosome banding patterns. The localization of the GPI locus to the same site on chromosomes with almost similar banding patterns suggests high degree of homology at these sites in the three species. Correlation between banding homologies and possible similarities at the molecular level is discussed.

Clonal relationship between Hashimoto thyroiditis and thyroid lymphoma

Background:Although Hashimoto thyroiditis (HT) is a predisposing factor for B-lineage thyroid lymphoma, clonal B-cell populations in HT are rare.Aim:To investigate whether there is a clonal relationship between HT and primary...

Homology of ciliary bands in Spiralian Trochophores

A number of hypotheses have been presented regarding the origins of the metazoans and, more specifically, the Bilateria. Using various phylogenetic analyses, characteristics have been mapped on phylogenetic trees to infer ancestral body plans and life history strategies of those ancestors. Many arguments on the evolution of the Bilateria are based on the presumed homology of certain characteristics of extant larva and adults, including various ciliated bands involved in feeding and locomotion. This article considers a recent study indicating that the second, downstream-collecting, ciliated band in the veliger larva of the gastropod mollusc, Crepidula fornicata, is actually derived from secondary trochoblasts (derived from second quartet micromeres), that normally form part of the prototrochal band found in other spiralian phyla (Hejnol et al. 2007). Despite previous arguments, these new findings suggest that the second ciliated band in the veliger larva is not homologous to the metatroch found in the trochophore larva of some other spiralians, such as the annelid, Polygordius lacteus. In the latter case, the metatroch was reported to be formed by a different set of lineage precursors (derived from third quartet micromeres) (Woltereck 1904). These findings have important implications for the interpretation of various hypotheses related to the evolution of metazoan phyla.

Conservation of aphidicolin-induced fragile sites in Papionini (Primates) species and humans

Fragile sites are considered structural features of mammalian chromosomes and a commonly repeated hypothesis is that they are evolutionarily conserved. We tested this hypothesis by establishing the subchromosomal homology of regions harbouring fragile sites in the chromosomes of humans, Macaca fascicularis (MFA) and Mandrillus sphinx (MSP). We delineated the interspecific homology of chromosome bands expressing aphidicolin-induced fragile sites of homologues to human chromosomes 1, 3, 5, 7, 18 and X by the comparative FISH of human BAC and YAC clones. Notably, two YAC clones known to span human chromosome regions containing fragile sites were shown to also span fragile sites in macaques and mandrills. The present comparative BAC/YAC mapping data represent, up to now, the most precise evidence of fragile site conservation during primate evolution.

Synteny assignment of four genes and two microsatellite markers in river buffalo (Bubalus bubalis L.)

Synteny assignment of four genes and two microsatellite markers in river buffalo (<i>Bubalus bubalis</i> L.)

Assignment of new loci to river buffalo chromosomes confirms the nature of chromosomes 4 and 5

Introduction Rapid development of the river buffalo physical map can be achieved by coupling its development to that of the cattle gene map. Syntenic conservation between cattle and buffalo has been demonstrated, mainly using somatic cell hybrids (de Hondt et al. 1991; El Nahas et al. 1993, 1996, 1998; de Hondt et al. 1997; El Nahta 1996; Oraby et al. 1977), and by using in situ hybridization as reviewed by Iannuzzi (1997).G‐ and R‐banding comparisons between cattle (2n = 60) and river buffalo (2n = 50) chromosomes have revealed a large number of banding homologies between the two species, both at early‐metaphase (Gupta and Ray‐Chaudhury 1978; Di Berardino et al. 1981) and prometaphase stages (Iannuzzi et al. 1990). Banding homology indicates that the five river buffalo biarmed pairs originate from centric fusion translocation between two of ten homologous cattle autosomes, which is very supportive of the hypothesis that both species have a common ancestor (Wurster and Benirschke 1968). Based on cytological analysis and banding homology between cattle and buffalo chromosomes, the five biarmed chromosomes of the river buffalo BBU1, BBU2, BBU3, BBU4, BBU5 were thought to originate from fusion of cattle chromosome (BTA) 1/25; 2/23; 8/19; 5/28; and 16/29 respectively (Iannuzzi et al. 1990; Report of the Committee for the Standardization of Banded Karyotopes of the River Buffalo 1994). However, the analysis of synteny between molecular markers assigned to different cattle syntenic groups demonstrated that BBU1 results from fusion of BTA 1 and 27 rather than 1 and 25 (El Nahas et al. 1977). This called for expanding the analysis of syntenic relationships between marker loci to confirm the nature of the other biarmed buffalo chromosomes.The purpose of this study is to test synteny between markers in buffalo and to confirm the nature of the biarmed buffalo chromosomes 4 and 5, using marker loci and somatic cell hybrids.

High resolution RBA-banding comparison between early prometaphase chromosomes of cattle (Bos taurus L.) and goat (Capra hircus L.) at 700 band level

High resolution RBA-banded early prometaphase chromosomes of cattle (Bos taurus L.) and goat (Capra hircus L.), from thymidine synchronized lymphocyte cultures, are compared at a level of 700 bands per haploid genome, with the purpose of detecting the extent of banding homologies between the two species and improving the resolution level of the ISCNDA (1989) standardized RBA-banded karyotypes. The results demonstrate that, at this level of resolution, at least 10 autosomes can be fully homologized between the two species, namely chromosomes 11, 16, 17, 18, 20, 21, 22, 23, 24 and 26, whereas the remaining autosomes show variations in some regions which require further investigations on more elongated chromosomes. Such investigations should also involve the use of GTG, GBG and RBG-banding techniques. These variations concern basically the relative distance of the bands from the centromeres or telomeres, appearance of subbands, and clustering of some positive bands.

Use of molecular markers for the identification of River Buffalo chromosomes: chromosome one

A standard karyotype for the River Buffalo has recently been established. The largest five chromosomes are biarmed and, based on the banding homology between cattle and buffalo chromosomes, were suggested to originate from the fusion of cattle acrocentric chromosomes. The origin of buffalo chromosome 1 is controversial due to the difficulty in differentiating between the small acrocentric cattle chromosomes. Using molecular markers assigned to cattle chromosomes, synteny between CD18, a marker for BTA1, and markers for small acrocentric cattle chromosomes BTA 24 to BTA 29 was investigated in buffalo/hamster somatic cell hybrids. The investigation revealed that CD18 is syntenic with ANT1, a marker for cattle chromosome 27. The present results confirm that buffalo BBU1 results from fusion of cattle BTA 1 and BTA 27. They also underline the importance of biarmed buffalo chromosomes for the identification of small cattle acrocentrics.

Synteny mapping in river buffalo.

The cosegregation of ten coding loci has been investigated, in a panel of 37 somatic cell hybrids resulting from the fusion of a hamster cell line and river buffalo lymphocytes, by use of Southern hybridization technique. Five syntenic groups, TCRB-PGY3, ASS-ABL, FUCA1P-CRYG, MBP-YES1, and CGN1-ACTA1, previously assigned to cattle as U13, U16, U17, U28, and U29 respectively, were also found to be syntenic in buffalo. Based on the extensive syntenic conservation and banding homology between cattle and river buffalo, comparative mapping predicts the localization of these syntenic groups on river buffalo Chromosomes (Chrs) :BBU7, BBU12, BBU2q, BBU22, and BBU4q respectively as they have been previously localized on cattle Chrs BTA4, BTA11, BTA2, BTA24 & BTA28.

Chromosomal evolution in bovids: a comparison of cattle, sheep and goat G- and R-banded chromosomes and cytogenetic divergences among cattle, goat and river buffalo sex chromosomes

A G- and R-banding comparison of cattle (Bos taurus, 2n = 60), goat (Capra hircus, 2n = 60) and sheep (Ovis aries, 2n = 54) chromosomes at the 450 band level was made. The study revealed a large number of banding homologies among the autosomes of the three species and resolved some ambiguities in arranging some of their small disputed acrocentrics by direct and indirect comparisons with some bovid marker chromosomes. A loss of the subcentromeric G-positive band in sheep chromosome 2g was observed when the G-banding patterns of sheep 2q and homologous cattle and goat chromosome 2 were compared. The chromosomal divergences among cattle, goat and river buffalo (Bubalus bubalis, 2n = 50) sex chromosomes are shown to have occurred by pericentric and paracentric inversions with a loss (or acquisition of constitutive heterochromatin.

Regional localization of the bovine interleukin-2 (IL2) gene to chromosome 17q22→q23 by in situ hybridization

The interleukin-2 (IL2) gene was localized to the q22-->q23 bands of chromosome 17 in cattle using radioactive in situ hybridization. The localization confirms the recent provisional assignment of syntenic group U23 to this bovine chromosome. Increasing evidence for chromosomal banding and gene location homology within the Bovidae suggests that the IL2 gene most likely also maps to chromosome 17 in sheep, goats and buffaloes.