Sequence Analysis Of Control Region Research Articles

Polyomavirus JC reactivation and noncoding control region sequence analysis in pediatric Crohn's disease patients treated with infliximab

The recent introduction of monoclonal antibodies in Crohn's disease (CD) management has been associated with the development of serious complications, such as the progressive multifocal leukoencephalopathy (PML), caused by JC polyomavirus (JCV) reactivation. Therefore, the aims of our study have been the investigation of the possible JCV reactivation in pediatric CD patients treated or not with infliximab, performing quantitative PCR in urine, plasma, and intestinal biopsies at the time of recruitment (t0) and every 4months in 1year of follow-up (t1, t2, and t3), and the analysis of the JCV noncoding control region (NCCR) to detect cellular transcription factors binding site mutations. Results obtained showed that, in urine and ileal specimens, JCV load significantly increased in infliximab-treated patients after 1year of treatment (t3), while viremia was significantly higher at t1. JCV NCCR sequence analysis showed a structure similar to CY archetype in 65/80 analyzed sequences, but the remaining 15/80, obtained exclusively from plasma and biopsies, evidenced a CY NCCR organization with two recurrent nucleotide changes, the 37-T to G transversion in box A Spi-B binding site and the 217-G to A transition in box F, and a box D deletion. These rearrangements were always found at t3 within seven infliximab-treated CD patients, who presented a very severe disease at t0. We can conclude that our rearranged NCCR sequences could be considered a marker of JCV virulence during mAb treatment, although none of our examined patients developed PML, and further studies on a larger cohort of patients should be performed.

Microsatellite variation and significant population genetic structure of endangered finless porpoises (Neophocaena phocaenoides) in Chinese coastal waters and the Yangtze River

The finless porpoise (Neophocaena phocaenoides) inhabits a wide range of tropical and temperate waters of the Indo-Pacific region. Genetic structure of finless porpoises in Chinese waters in three regions (Yangtze River, Yellow Sea, and South China Sea) was analyzed, including the Yangtze finless porpoise which is widely known because of its highly endangered status and unusual adaptation to freshwater. To assist in conservation and management of this species, ten microsatellite loci were used to genotype 125 individuals from the three regions. Contrary to the low genetic diversity revealed in previous mtDNA control region sequence analyses, relatively high levels of genetic variation in microsatellite profiles (H E = 0.732–0.795) were found. Bayesian clustering analysis suggested that finless porpoises in Chinese waters could be described as three distinct genetic groups, which corresponded well to population “units” (populations, subspecies, or species) delimited in earlier studies, based on morphological variation, distribution, and genetic analyses. Genetic differentiation between regions was significant, with F ST values ranging from 0.07 to 0.137. Immigration rates estimated using a Bayesian method and population ancestry analyses suggested no or very limited gene flow among regional types, even in the area of overlap between types. These results strongly support the classification of porpoises in these regions into distinct evolutionarily significant units, including at least two separate species, and therefore they should be treated as different management units in the design and implementation of conservation programmes.

High levels of mitochondrial DNA divergence within short-eared rock-wallaby (Petrogale brachyotis) populations in northern Australia

Most population genetics studies of rock-wallabies conducted to date have examined remnant colonies of threatened species inhabiting southern Australia. In this study we examined the natural pattern of contemporary and long-term gene flow among colonies of the widespread and abundant short-eared rock-wallaby, Petrogale brachyotis, in the relatively unmodified landscapes of Australia’s tropical north. We sampled 105 wallabies from seven colonies 1.2 km to 250 km apart. Mitochondrial DNA (mtDNA) control region sequence analysis was conducted on samples from all colonies and microsatellite analysis (10 loci) on samples from the three largest colonies. The microsatellite data revealed no evidence of inbreeding within colonies, but higher levels of genetic diversity were found in the Kakadu National Park population compared with the smaller, more isolated colonies at Litchfield National Park. Both the mtDNA and microsatellite results showed that populations of P. brachyotis are naturally highly structured even within this relatively intact landscape, with only limited contemporary and long-term gene flow between colonies more than 1.2 km apart. Nine mtDNA control region haplotypes were identified within the seven colonies. There were unusually high levels of sequence divergence (up to 6.9%) within colonies at Litchfield NP. This divergence suggests that multiple taxa may exist within what is currently recognised as P. brachyotis. Alternatively, if current taxonomy is correct, the high levels of divergence raise the possibility of ancestral isolation and divergence of populations in allopatry with subsequent admixture at a secondary contact zone. The possibility that these unusually divergent haplotypes result from introgressive interspecific hybridisation with the sympatric P. concinna appears unlikely.

Genetic characterization of loggerhead turtle (Caretta caretta) individuals stranded and caught as bycatch from the North-Central Adriatic Sea

AbstractThe North Adriatic Sea is considered a critical feeding and developmental area for Mediterranean loggerhead turtles. In this study, a comparative analysis of mitochondrial DNA control region sequences was carried out on sixty-five loggerhead individuals stranded and caught as bycatch in the Adriatic Sea from 1999 to 2002. We demonstrated the existence of genetic relationships between the North-Central Adriatic (NCA) aggregate and the Central-Eastern Mediterranean rookeries. Short-range and long-range migrations towards the Adriatic Sea were hypothesized by the finding of individuals bearing haplotypes endemic to the nesting populations of Greece and Turkey. The occurrence of individuals belonging to dimensional classes from juveniles to mature adults strongly support both the feeding and developmental role of the NCA.

Genetic variation and evolutionary demography of Fenneropenaeus chinensis populations, as revealed by the analysis of mitochondrial control region sequences

Genetic variation and evolutionary demography of the shrimp Fenneropenaeus chinensis were investigated using sequence data of the complete mitochondrial control region (CR). Fragments of 993 bp of the CR were sequenced for 93 individuals from five localities over most of the species' range in the Yellow Sea and the Bohai Sea. There were 84 variable sites defining 68 haplotypes. Haplotype diversity levels were very high (0.95 ± 0.03-0.99 ± 0.02) in F. chinensis populations, whereas those of nucleotide diversity were moderate to low (0.66 ± 0.36%-0.84 ± 0.46%). Analysis of molecular variance and conventional population statistics (FST ) revealed no significant genetic structure throughout the range of F. chinensis. Mismatch distribution, estimates of population parameters and neutrality tests revealed that the significant fluctuations and shallow coalescence of mtDNA genealogies observed were coincident with estimated demographic parameters and neutrality tests, in implying important past-population size fluctuations or range expansion. Isolation with Migration (IM) coalescence results suggest that F. chinensis, distributed along the coasts of northern China and the Korean Peninsula (about 1000 km apart), diverged recently, the estimated time-split being 12,800 (7,400-18,600) years ago.

Iranian samples of brown trout Salmo trutta or rainbow trout Oncorhynchus mykiss: Comparison of evidence from allozyme and mitochondrial DNA control region sequence analysis

Iranian samples of brown trout Salmo trutta or rainbow trout Oncorhynchus mykiss: Comparison of evidence from allozyme and mitochondrial DNA control region sequence analysis

Mitochondrial DNA control region sequence analysis of Mataco–Guaicurú speaking tribes from Argentina

In this work we analyze three Mataco–Guaicurú speaking Amerindian tribes that inhabit the Northern Argentinean portion of Gran Chaco region. One hundred and sixty-eight samples of unrelated males belonging to two linguistic families: Guaycurú (Toba from Chaco, n = 27; Toba from Formosa, n = 37 and Pilaga from Formosa, n = 56) and Mataco (Wichi from Formosa, n = 48) were investigated. The entire Control Region of the mtDNA from position 16024 to 576 was sequenced. EMPOP sequencing strategy was employed including the use of 10 primers for each sample. Specific haplotypes were found in these populations with very high frequency. These findings could provide clues to address the ethnicity of a sample in routine forensic casework.

Amerindian mitochondrial DNA haplogroups predominate in the population of Argentina: towards a first nationwide forensic mitochondrial DNA sequence database

The study presents South American mitochondrial DNA (mtDNA) data from selected north (N = 98), central (N = 193) and south (N = 47) Argentinean populations. Sequence analysis of the complete mtDNA control region (CR, 16024-576) resulted in 288 unique haplotypes ignoring C-insertions around positions 16193, 309, and 573; the additional analysis of coding region single nucleotide polymorphisms enabled a fine classification of the described lineages. The Amerindian haplogroups were most frequent in the north and south representing more than 60% of the sequences. A slightly different situation was observed in central Argentina where the Amerindian haplogroups represented less than 50%, and the European contribution was more relevant. Particular clades of the Amerindian subhaplogroups turned out to be nearly region-specific. A minor contribution of African lineages was observed throughout the country. This comprehensive admixture of worldwide mtDNA lineages and the regional specificity of certain clades in the Argentinean population underscore the necessity of carefully selecting regional samples in order to develop a nationwide mtDNA database for forensic and anthropological purposes. The mtDNA sequencing and analysis were performed under EMPOP guidelines in order to attain high quality for the mtDNA database.

Multilocus assignment analyses reveal multiple units and rare migration events in the recently expanded yellow‐eyed penguin (Megadyptes antipodes)

The identification of demographically independent populations and the recognition of management units have been greatly facilitated by the continuing advances in genetic tools. Managements units now play a key role in short-term conservation management programmes of declining species, but their importance in expanding populations receives comparatively little attention. The endangered yellow-eyed penguin (Megadyptes antipodes) expanded its range from the subantarctic to New Zealand's South Island a few hundred years ago and this new population now represents almost half of the species' total census size. This dramatic expansion attests to M. antipodes' high dispersal abilities and suggests the species is likely to constitute a single demographic population. Here we test this hypothesis of panmixia by investigating genetic differentiation and levels of gene flow among penguin breeding areas using 12 autosomal microsatellite loci along with mitochondrial control region sequence analyses for 350 individuals. Contrary to our hypothesis, however, the analyses reveal two genetically and geographically distinct assemblages: South Island vs. subantarctic populations. Using assignment tests, we recognize just two first-generation migrants between these populations (corresponding to a migration rate of < 2%), indicating that ongoing levels of long-distance migration are low. Furthermore, the South Island population has low genetic variability compared to the subantarctic population. These results suggest that the South Island population was founded by only a small number of individuals, and that subsequent levels of gene flow have remained low. The demographic independence of the two populations warrants their designation as distinct management units and conservation efforts should be adjusted accordingly to protect both populations.

Genetic population structure of the Swordfish ( Xiphias gladius) in the southwest Indian Ocean: Sex-biased differentiation, congruency between markers and its incidence in a way of stock assessment

Genetic variation was surveyed at 11 microsatellite loci and at 517 bp of the mitochondrial control region to investigate the presence of genetic stock structure in swordfish ( Xiphias gladius) in four proximal localities of the southwest Indian Ocean. One aim of this study was to serve as a preliminary examination for congruency of structure detected by these two genetic markers, prior to conducting a more comprehensive basin-wide survey of the Indian Ocean and nearby surrounding areas. Analyses of multilocus microsatellite genotypes and mitochondrial control region sequences both revealed a great homogeneity between samples. Genetic diversity detected at the regional scale was not significantly higher than detected at the local scale. Results suggest that the southwest Indian Ocean globally functions as a unique panmictic population. However, some discrete genetic differences appeared that could possibly indicate influence from a second genetic pool in the northern part of the Indian Ocean. This structure appeared to be sex-dependent with genetic differences higher among female than among male samples. This result may indicate a higher level of spawning area fidelity for females with a subsequent sampling bias tending to homogenise male genotypic distributions.

Mitochondrial control region sequence analyses indicate dispersal from the US East Coast as the source of the invasive Indo-Pacific lionfish Pterois volitans in the Bahamas

LionWsh are popular aquarium Wsh from the Indo-PaciWc that have invaded the western Atlantic. Two species, Pterois volitans and P. miles, were well established along the United States east coast before the Wrst lionWsh were reported from the Bahamas in 2004, where they quickly dispersed throughout the archipelago by 2007. The source of the Bahamian lionWsh invasion has been in ques- tion because of the hypothesized low connectivity between Florida and Bahamas reef species as well as the temporal lag in their arrival in the Bahamas. Mitochondrial control region haplotypes (680 bp) were determined and analyzed for lionWsh specimens from the Bahamas, North Carolina, and two sites within their native range (Indonesia and the Philippines). Exact tests, pairwise F st and AMOVA analy- ses all showed no signiWcant diVerentiation between the Bahamas and North Carolina specimens. The similarity between the Bahamas and North Carolina lionWsh was also reXected in a minimum spanning network and neighbor- joining distance tree generated from the data. Sequence analyses also revealed the presence of only Pterois volitans, as no P. miles were detected in the Bahamian sample. These results indicate that the source of the Bahamian lion- Wsh is egg and larval dispersal from the United States east coast population, and support previous models of reef Wsh dispersal that suggest a low level of connectivity between the Bahamas and east coast of Florida.

Delineation of discrete population segments of shortnose sturgeon Acipenser brevirostrum based on mitochondrial DNA control region sequence analysis

Shortnose sturgeon Acipenser brevirostrum is federally listed as ‘‘an endangered species threatened with extinction’’ in the U.S. but its listing status is currently under review. As part of this process, the U.S. National Marine Fisheries Service will determine if shortnose sturgeon are divided into Distinct Population Segments (DPS) across its distribution. In this regard, we sought to determine if shortnose sturgeon occur in genetically “discrete population segments,” and if so, the boundaries of each. We used mitochondrial DNA (mtDNA) control region sequence analysis to assess the genetic discreteness of 14 of 19 river populations that were recommended as DPS in the 1998 Final Recovery Plan for Shortnose Sturgeon. Nine of the 14 proposed DPS proved significantly discrete (P < 0.05 after Bonferoni correction) from both of their bracketing populations, the exceptions being those in the Penobscot River, Chesapeake Bay, Cooper River, and Ogeechee River (our sample from the Cape Fear River was insufficient to statistically analyze). Haplotype frequencies in the newly “rediscovered” Penobscot River collection were almost identical to those in the proximal Kennebec River system. Genetic data in combination with tagging results suggest that shortnose sturgeon in the Penobscot River are probably migrants from the Kennebec. Likewise, shortnose sturgeon found today within the Chesapeake Bay appear to be migrants from the Delaware River. While haplotype frequencies in the remnant Santee River population in Lake Marion differed significantly from those in nearby Winyah Bay, they did not differ significantly from those in the Cooper River. This suggests that the Cooper River harbors descendants of the Santee River population that are unable to access their historical spawning locales. The Ogeechee River collection was not genetically distinct from that in the nearby Savannah River, suggesting that it may host descendants of hatchery-reared individuals of Savannah River ancestry. Our genetic results indicate that most, but not all, rivers with shortnose sturgeon host genetically discrete populations, constituting important information in the consideration of DPS designations. However, shortnose sturgeon migrations through coastal waters to proximal rivers and release of hatchery-reared fish may confound results from genetic studies such as ours and lead to the possible misidentification of discrete population segments.

MtDNAmanager: a Web-based tool for the management and quality analysis of mitochondrial DNA control-region sequences

BackgroundFor the past few years, scientific controversy has surrounded the large number of errors in forensic and literature mitochondrial DNA (mtDNA) data. However, recent research has shown that using mtDNA phylogeny and referring to known mtDNA haplotypes can be useful for checking the quality of sequence data.ResultsWe developed a Web-based bioinformatics resource "mtDNAmanager" that offers a convenient interface supporting the management and quality analysis of mtDNA sequence data. The mtDNAmanager performs computations on mtDNA control-region sequences to estimate the most-probable mtDNA haplogroups and retrieves similar sequences from a selected database. By the phased designation of the most-probable haplogroups (both expected and estimated haplogroups), mtDNAmanager enables users to systematically detect errors whilst allowing for confirmation of the presence of clear key diagnostic mutations and accompanying mutations. The query tools of mtDNAmanager also facilitate database screening with two options of "match" and "include the queried nucleotide polymorphism". In addition, mtDNAmanager provides Web interfaces for users to manage and analyse their own data in batch mode.ConclusionThe mtDNAmanager will provide systematic routines for mtDNA sequence data management and analysis via easily accessible Web interfaces, and thus should be very useful for population, medical and forensic studies that employ mtDNA analysis. mtDNAmanager can be accessed at .

Insights on common dolphin (Delphinus delphis) social organization from genetic analysis of a mass-stranded pod

Compared to terrestrial mammals, little is known of cetacean social systems as they are generally less accessible to behavioral investigations due to their aquatic environment. The present study investigates group structure of the pelagic common dolphin, Delphinus delphis, using genetic markers. Tissue samples from 52 individuals representing a recent live mass-stranding event were compared to 42 single strandings taken from presumably different groups. The mass-stranding event occurred in 2002 on the French coast of the English Channel, whereas the single strandings were collected between 1993 and 2003 along the western coast of France (Bay of Biscay and English Channel). Analysis of mitochondrial DNA control region sequences indicated that genetic variability within the mass-stranded pod was similar to variability observed in single strandings. The mass-stranded group was composed of 41 different mitochondrial haplotypes or matrilines while the single strandings revealed 29 different haplotypes. Analysis of 11 microsatellite loci revealed that average relatedness of the mass-stranded pod was not different from average relatedness among all single strandings suggesting that individuals within the group had no closer kin relationships than animals taken from presumably different groups. These results do not support a matriarchal system and suggest that common dolphins constituting a pod are not necessarily genetically related.

Population structure of striped marlin (Kajikia audax) in the Pacific Ocean based on analysis of microsatellite and mitochondrial DNA

Genetic variation was surveyed at five microsatellite loci and the mitochondrial control region (819 bp) to test for the presence of genetic stock structure in striped marlin ( Kajikia audax ) collections taken from seven locations throughout the Pacific Ocean. Temporal replicates separated by 9 years were taken off Japan, and three temporal samples spanning 11 years were collected off the coast of eastern Australia. Analyses of multilocus microsatellite genotypes and mitochondrial control region sequences showed no significant heterogeneity among collections taken from the same location in different years; however, significant spatial genetic heterogeneity was observed across all samples for microsatellite markers (FST= 0.013, P &lt; 0.001). Mitochondrial control region sequences were not different across all samples (ΦST= –0.01, P = 0.642). Analyses of molecular variance (AMOVA) revealed significant genetic differentiation between geographic regions for both microsatellite and mitochondrial markers. These results suggest the presence of genetically discrete populations within the Pacific Ocean and are supported by both the results of tagging studies, which show limited dispersal, and the presence of geographically separated spawning grounds.

‘Mitominis’: multiplex PCR analysis of reduced size amplicons for compound sequence analysis of the entire mtDNA control region in highly degraded samples

The traditional protocol for forensic mitochondrial DNA (mtDNA) analyses involves the amplification and sequencing of the two hypervariable segments HVS-I and HVS-II of the mtDNA control region. The primers usually span fragment sizes of 300-400 bp each region, which may result in weak or failed amplification in highly degraded samples. Here we introduce an improved and more stable approach using shortened amplicons in the fragment range between 144 and 237 bp. Ten such amplicons were required to produce overlapping fragments that cover the entire human mtDNA control region. These were co-amplified in two multiplex polymerase chain reactions and sequenced with the individual amplification primers. The primers were carefully selected to minimize binding on homoplasic and haplogroup-specific sites that would otherwise result in loss of amplification due to mis-priming. The multiplexes have successfully been applied to ancient and forensic samples such as bones and teeth that showed a high degree of degradation.

Latent human polyomavirus infection in pregnancy: investigation of possible transplacental transmission

The purpose of the study was to investigate the transplacental transmission of the human polyomaviruses JCV and BKV. Urine and blood samples from 300 pregnant women underwent cytological analysis to search for 'decoy cells', nested PCR to identify presence and genotype of isolated polyomaviruses, and sequence analysis of the transcription control region. Nested PCR was also used to study the umbilical cord blood of all their newborns. Decoy cells were identified in only one urine sample (1/300; 0.33%); polyomavirus DNA was detected in 80 urine samples (26.6%) corresponding to BKV alone in 28 samples (9.3%), JCV alone in 49 samples (16.3%) and both JCV-BKV in three samples (1%). Blood samples were positive in 17 cases (5.6%), corresponding to BKV alone in 10 (3.3%), and JCV alone in 7 (2.3%). Rearrangements of the transcription control region were found in only one urinary JCV strain, consisting of the insertion of 13 bp at D block, whereas point mutations were identified in 11 BKV and 11 JCV strains detected from urine. Sequence analysis of the BKV strains detected in blood samples revealed a 20 bp insertion of P block (P42-61) in human chromosomes 20 (five cases) and 14 (three cases); two JCV strains had single bp point mutations. The search for polyomavirus DNA in umbilical cord blood samples was always negative. Polyomavirus DNA was frequently detected in pregnancy, whereas genomic rearrangements were rare, and no evidence of transplacental transmission of polyomavirus was obtained.

Molecular variability in Brycon cf. pesu Muller and Troschel, 1845 (Characiformes: Characidae) from the Araguaia-Tocantins Basin

Brycon pesu is a small-sized fish distributed throughout the Amazon and Orinoco Basins and other coastal basins of northeastern South America. Brycon cf. pesu specimens from the Araguaia-Tocantins Basin are currently separated into two morphotypes, Brycon sp1 and Brycon sp2, owing to different coloration of their anal fin. Brycon sp2 has a reddish margin stripe on the anal fin which morphologically distinguishes it from Brycon sp1. In the present research, nuclear and mitochondrial markers were used to test the hypothesis that the Brycon sp1 and Brycon sp2 morphotypes are distinct species. Specimens from the two morphotypes were collected from the Lajeado Hydroelectric Plant and the Palmas River in the Araguaia-Tocantins Basin. Thirty-five loci obtained by the amplification of five inter-simple sequence repeat primers were analyzed but no species-specific bands were detected. Electrophoretic profiles obtained from 5S rDNA non-transcribed spacer amplification failed to show any differentiation in morphotypes. These results were corroborated by nucleotide sequence analysis of the mtDNA control region, in which 24 polymorphic nucleotide sites, representing a polymorphism rate of only 5%, were detected. The low rates of polymorphism detected by inter-simple sequence repeat, non-transcribed spacer and mtDNA D-loop markers strongly reject the hypothesis that the two morphotypes Brycon sp1 and Brycon sp2 represent distinct species within Brycon cf. pesu. Further studies are needed to obtain conclusive data on the notion that the coloration of the anal fin is an intraspecific polymorphism, possibly related to environmental factors.

Genetic stock structure of blue-eye trevalla (Hyperoglyphe antarctica) and warehous (Seriolella brama and Seriolella punctata) in south-eastern Australian waters

Blue-eye trevalla (Hyperoglyphe antarctica), blue warehou (Seriolella brama) and silver warehou (Seriolella punctata) from the family Centrolophidae are three commercially important species in the Australian fishery. These species are currently managed as single stocks. We tested the hypothesis that patterns of phenotypic structuring in these species reflect underlying genetic stock structure using an analysis of mitochondrial DNA control region sequences. The analysis revealed high levels of haplotype diversity within populations. The most common haplotypes for the species occurred in all geographical locations sampled. For S. brama, although structuring was not significant after Bonferroni correction, differences between two sites were sufficient to warrant caution in the management of fishery zones for this species. There were also some indications of structuring when sites were grouped into common regions. Demographic analysis suggested that S. brama might have had a history of population bottlenecks followed by sudden population expansion, potentially contributing to genetic structuring in the fishery. No structuring was detected for H. antarctica and S. punctata. The present study highlights the need for, and the utility of, multiple sources of information, that is, genetic, phenotypic, behavioural and ecological, when managing marine fisheries and the need to take a cautionary approach to the interpretation of genetic data for fisheries management.

Human papillomavirus genotypes and HPV16 variants in penile carcinoma

The causative role of human papillomaviruses (HPV) and HPV16 variants has been extensively studied in uterine cervix dysplastic lesions and invasive carcinoma; few such studies, however, have been performed in penile tumors. We have investigated HPV genotype and HPV16 variant distribution on 41 penile cancer biopsies from Italian patients. Cases were extracted from the respective pathology departments databases of National Cancer Institutes in Naples and Milan. HPV sequences were detected by PCR and characterized by direct sequence analysis. Among the 19 HPV-positive cases (46.3%) 2 viral genotypes were identified (HPV16 and 18) with HPV16 accounting for 94.7% (18 out of 19) of the infections. Sequence analysis of E6, E7 genes and long control region (LCR) of 18 HPV16 isolates allowed the identification of European (E-G-350) and non-European (AA and Af-1) variants in 44.4% and in 55.6% of the samples, respectively. The AA variant alone represented 44.4% of all HPV16 infections, a significantly higher frequency of that observed in cervical carcinoma from Italian women (Tornesello et al., J Med Virol 2004;74:117-26). Our results suggest that HPV16 has a very high prevalence among penile cancer patients in Italy and the increased frequency of HPV16 non-European classes, particularly the AA, suggests that they are more oncogenic than European variants in penile tissue.