HVI Region Research Articles

Inactivation of cerebellar output axons impairs acquisition of conditioned eyeblinks

Acquisition of classically conditioned eyeblink responses (CRs) in the rabbit critically depends on intermediate cerebellum-related neural circuits. A highly efficient method for determining possible sites of plasticity within eyeblink circuits is the reversible inactivation of circuit components during learning. Inactivation of either the HVI region of the cerebellar cortex or the cerebellar interposed nuclei (IN) during learning is known to prevent CR acquisition. In contrast, inactivating cerebellar efferent axons in the brachium conjunctivum (BC) with small injections of tetrodotoxin (TTX) has been reported to have no effect on CR acquisition. This suggested that the intermediate cerebellum is essential for learning CRs and that activity mediated by the BC is not required for this process. Since we previously found that BC inactivation blocks CR extinction we re-examined its role in CR acquisition. To ensure complete and long-lasting inactivation of the BC, we injected before each training session doses of TTX that were larger than those in the previous acquisition study. Contrary to the previous negative findings, we found that this temporary block of axons in the brachium conjunctivum prevented normal acquisition of CRs. Injecting TTX directly in the adjacent lateral lemniscus, which could possibly influence CR acquisition, had no effect on learning. In addition, a functional test of TTX diffusion around the BC indicated that the inactivation did not affect other known parts of eyeblink circuits, such as the cerebellar interposed nuclei, the middle cerebellar peduncle or the contralateral red nucleus. We conclude that this form of associative learning in the rabbit eyeblink system requires extra-cerebellar learning and/or cerebellar learning that depends on the operation of cerebellar feedback loops.

Genetic variability and differentiation in Spanish roe deer ( Capreolus capreolus): A phylogeographic reassessment within the European framework

Here we study 109 Iberian roe deer individuals corresponding to 9 Spanish populations. Individuals were sampled at locations that are expected to have acted as refugia for the species during the 20th century. Samples were analyzed for a 436 bp mtDNA fragment from the HVI region of mitochondrial DNA and 10 microsatellites. The 109 sequences gave 31 different haplotypes that enabled identification of a new haplogroup (mainly present in Northwestern Iberian populations and representing roughly a third of our samples) that is not present in other European roe deer populations. Using microsatellites, correspondence analysis and molecular coancestry information revealed high molecular differentiation among Northwestern and Central-Southern Spain roe deer populations. Both sequence and microsatellite analysis reveal that the Spanish roe deer populations are genetically heterogeneous and have high genetic structure clearly separating the Pyrenean-reintroduced populations and two main areas for the species in Spain (Northwestern and Central-Southern) coinciding with the two main areas acting as refugia for the majority of the mammal species during glaciations. The implications of the obtained information as regards the phylogeography of the species are discussed together with suggestions as to appropriate strategies for the conservation and management of populations.

HVI and HVII sequence polymorphisms of the human mtDNA in the North of Portugal: Population data and maternal lineages

We have analysed the sequence polymorphisms of the hypervariable segments I and II (HVI and HVII) of the human mitochondrial DNA (mtDNA) in the population of North of Portugal. The aim of this study was to create a population database for HVI and HVII regions and to analyse these two segments in maternal relatives. For the population data, the HVI and HVII segments were analysed in 142 unrelated and healthy individuals, chosen randomly, from the North of Portugal. For the maternal lineages analysis, these two segments were studied in 24 sets of families from that region of Portugal. The sequence polymorphisms of the HVI and HVII were determined by polymerase chain reaction (PCR) and direct sequencing.

Analysis of the HVI, HVII and HVIII regions of mtDNA in 400 unrelated Japanese

Sequence polymorphism of the hypervariable regions HVI, HVII and HVIII of mitochondrial DNA (mtDNA) was analyzed in a sample of 400 unrelated Japanese individuals living in Gifu Prefecture (central region of Japan) by PCR amplification and direct sequencing. A total of 308 different haplotypes resulting from 187 polymorphic positions was found in our Japanese population sample. The most common haplotype was shared by 10 individuals. The genetic diversity and the genetic identity were calculated to be 0.9975 and 0.0050, respectively.

Length heteroplasmy in the HVI control region

The variability in np16184–16193 HVI region presents two different types of polymorphism: sequence variability and length variability with length heteroplasmy. It is important to define properly the HVI homopolimeric regions for a correct classification of homopolimeric track defining the number of As and Cs and the various population types identified by adjacent bases to the homopolimeric track.

Multi-substrata analysis on Siberian mummies: A different way for validation in ancient DNA studies?

Ancient DNA results are always submitted to caution due to the technical difficulties induced by the minute amounts, the degraded nature of the template and the high risk of contamination. A list of criteria of validation has been published as a guideline for ancient DNA researchers [[1] A. Cooper, H.N. Poinar, Ancient DNA: do it right or not at all, Science 289 (5482) (2000) 1139.]. In addition to these criteria, the analysis of different substrates, i.e. bone, teeth and hairs of the same individual, could be another way to ensure the reliability of the results. This study presents the data obtained on bones, molar teeth and hairs from two Siberian specimens dated from the 18th century. These two subjects excavated from frozen graves were mummified. Their exceptional state of preservation allowed us to test the amplification of autosomal and Y chromosomal STRs and of the HVI region on the three types of substrate. This approach allowed the identification of artefacts on STR profiles, common when working with Low Copy Number DNA. Indeed, the comparison of the profiles obtained from bones and teeth highlights allelic dropouts and spurious alleles for the bone samples. The possibility to compare results from different substrates, in spite of the limited number of possible cases, represents another, and interesting, criterion to confirm the authenticity of ancient DNA results.

The Origins of Iberian Horses Assessed via Mitochondrial DNA

Despite a number of recent studies that have focused on the origin of domestic horses, genetic relationships between major geographical clusters still remain poorly understood. In this study we analyzed a 296 bp mtDNA fragment from the HVI region of 171 horses representing 11 native Iberian, Barb, and Exmoor breeds to assess the maternal phylogeography of Iberian horses. The mtDNA haplogroup with a CCG motif (nucleotide position 15,494 to 15,496) was the most frequent in Iberian and Barb breeds (0.42 and 0.57, respectively), regardless of geographic location or group of breeds. This finding supports the close genetic relationship between the ancestral mare populations of the Iberian Peninsula and Northern Africa. Phenotypic differences among the Northern and Southern Iberian groups of breeds are not explained by population subdivision based on maternal lineages. Our results also suggest that Northern Iberian ponies--which are phenotypically close to British ponies, especially Exmoor--are the result of an introgression rather than population replacement.

MtDNA HVI length heteroplasmic profile in different tissues of maternally related members

Sequencing of the homopolymeric tract of cytosines (C-stretch) in human mitochondrial HVI region usually results in a blurred pattern beyond it when a T/C transition at nt 16189 occurs: it depends on a length heteroplasmy probably arising through a replication slippage. This study aims to investigate the distribution of heteroplasmic length variants within three related individuals along maternal lineage by cloning approach. Sequencing of multiple independent clones (12–14) is sufficient to yield heteroplasmic profiles. In addition, we illustrate a direct correlation between expansion of heteroplasmy modal length and reduction of the number of adenines preceding the homopolymeric tract; this association may be useful in pedigree analysis and in forensic field for tissues comparison, single hair sample included.

Analysis of the HVI and HVII regions of mitochondrial DNA in 100 individuals from North of Portugal

The analysis of mitochondrial DNA (mtDNA) control region is of great importance in forensic casework and evolution studies. The aim of this study was to create a population database for the HVI and HVII regions of mtDNA in the population of North of Portugal. The HVI and HVII segments were studied in 100 unrelated and healthy individuals by direct sequencing. Nucleotide substitutions were found using Anderson's reference sequence. Length and position heteroplasmy were observed. The genetic structure of the population was analysed. The match probability and discrimination power values were calculated. The phylogenetic analysis for HVI segment was made following classification into haplogroups and genetic distances were calculated. These analyses place the North of Portugal population among the European Caucasoid populations.

Measuring by fragment analysis the proportion of length variants in samples carrying length heteroplasmy at the homopolymeric C-stretch in mitochondrial HVII region

We describe a method to estimate the proportion of length variants in samples carrying mtDNA homopolymeric C-stretch (303 to 315) originated by a T310C transition. Sequencing analysis of these samples produces blurred electropherograms and the exact number of C-residues remains ambiguous. This has important consequences in forensic casework, since one of the aims in the interpretation of mtDNA profiles is the determination of the existence of a match or a mismatch between two or more sequences under study. HV-2B region (positions 172 to 408) was amplified with two sets of labelled primers. Electrophoresis of fluorescently labelled amplicons was performed on an ABI310 or ABI377. Results were reproducible in repeated PCR experiments, using the two sets of primers and using either capillary or slab-gel electrophoresis.

The EDNAP mitochondrial DNA population database (EMPOP) collaborative exercises: organisation, results and perspectives

This paper presents an overview of the organisation and the results of the collaborative exercises (CE) of the European DNA Profiling (EDNAP) Group’s mitochondrial DNA population database project (EMPOP). The aim of the collaborative exercises was to determine whether uniformity of mtDNA sequencing results could be achieved among different laboratories. These were asked to sequence either the complete mtDNA control region or the two hypervariable regions HVI (16024–16365) and HVII (73–340) from DNA extracts, buccal swabs or bloodstains, proceeding in accordance with the protocol and strategies used in each individual laboratory. The results of the collaborative exercises were employed to identify possible sources of errors that could arise during the analysis and interpretation of mtDNA profiles. These findings were taken as a basis to tentatively make suitable arrangements for the construction of a high quality mtDNA database. One hundred fifty mtDNA profiles were submitted to the evaluating laboratory, and disaccording profiles were classified into four groups corresponding to the source of error: clerical errors, sample mix-ups, contaminations and discrepancies with respect to the mtDNA nomenclature. Overall, 14 disaccording haplotypes (16 individual errors) were observed. The errors included 10 clerical errors, 3 interpretation problems, 2 cases of sample mix-up and 1 case of point heteroplasmic mixture, where the 2 sequencing reactions brought inconsistent base calls. This corresponds to an error rate of 10.7% in a virtual mtDNA database consisting of the collaborative exercise results. However, this estimate is still conservative compared to conclusions drawn by authors of meanwhile numerous publications critically reviewing published mtDNA population databases. Our results and earlier published concerns strongly emphasize the need for appropriate safety regulations when mtDNA profiles are compiled for database purposes in order to accomplish the high standard required for mtDNA databases that are used in the forensic context.

Sequence polymorphism of the mitochondrial DNA control region in the Slovenian population.

The forensic application of mitochondrial DNA (mtDNA) typing requires large and regionally well-defined databases. To expand the database for forensic identification purposes in Slovenia, the mtDNA control region sequences of the hypervariable regions HVI and HVII were determined in a population of 129 maternally unrelated Slovenians, using a fluorescent-based capillary electrophoresis sequencing method. A total of 111 different haplotypes resulting from 124 polymorphic positions (80 polymorphic positions in HVI and 44 in HVII) were found. Of these, 101 mtDNA types were unique, 6 haplotypes were shared by 2 individuals, 1 haplotype by 3 individuals, 2 haplotypes by 4 individuals, and the most common haplotype was found in 5 individuals. The most frequent haplotypes in the Slovenian population,263(G), 315.1(C) and 263(G), 309.1(C), 315.1(C) are also the most common in other European populations. The data support the concept that these haplotypes may represent a common European mtDNA sequence types. The sequence poymorphisms were compared to the databases of west Austria and central Italy and the HVI and HVII sequence matching probabilities within and between populations were calculated. It is 1.1-4.5 times more likely to find a sequence match in a random pair of Slovenians than in a random Slovenian-Italian pair and in a random Slovenian-Austrian pair. The length heteroplasmy in the homopolymeric C-stretch regions located at nucleotide positions 16184-16193 in HVI and at positions 303-315 in HVII was observed in 17% and 8% of individuals, respectively. A statistical estimate of the results for this population showed the random match probability and the genetic diversity of 1.16% and 0.996, respectively.

Sequence polymorphisms of the mitochondrial DNA control region and phylogenetic analysis of mtDNA lineages in the Japanese population.

Sequence polymorphisms of the hypervariable mitochondrial DNA (mtDNA) regions HVI and HVII, and coding region polymorphisms were investigated in 211 unrelated individuals from the Japanese population. Sequence comparison of the HVI and HVII regions led to the identification of 169 mitochondrial haplotypes defined by 147 variable positions. Among them 145 types were observed in only 1 individual; the other 24 types were shared by 2 or more individuals. The gene diversity was estimated at 0.9961, and the probability of two randomly selected individuals from the population having identical mtDNA types was 0.86%. We also established phylogenetic haplogroups in the Japanese population based on the coding and control region polymorphisms and compared the haplotypes with those in other Japanese, Korean and Chinese populations. As a result, three new subhaplogroups, G4a, G4b, and N9b, and several haplotypes specific for the Japanese and Korean populations were identified. The present database can be used not only for personal identification but also as an aid for geographic or phenotype (race) estimation in forensic casework in Japan.

Different informativeness of the three hypervariable mitochondrial DNA regions in the population of Bologna (Italy)

Mitochondrial DNA (mtDNA) sequence variations at hypervariable regions HVI, HVII and HVIII were analysed in 100 unrelated Italians from Bologna. The values of the statistical parameters are in agreement with the range of European populations. We suggest that the less informative HVIII region may be useful to distinguish HVI–HVII identical sequences in forensic analysis especially when nuclear DNA cannot be investigated.

Maternal and paternal lineages of the Samaritan isolate: mutation rates and time to most recent common male ancestor.

The Samaritan community is a small, isolated, and highly endogamous group numbering some 650 members who have maintained extensive genealogical records for the past 13-15 generations. We performed mutation detection experiments on mitochondrial DNAs and Y chromosomes from confirmed maternal and paternal lineages to estimate mutation rates in these two haploid compartments of the genome. One hundred and twenty four DNA samples from different pedigrees (representing 200 generation links) were analyzed for the mtDNA hypervariable I and II regions, and 74 male samples (comprising 139 links) were typed for 12 Y-STRs mapping to the non-recombining portion of the Y chromosome (NRY). Excluding two somatic heteroplasmic substitutions and several length variants in the homopolymeric C run in the HVII region, no mutations were found in the Samaritans' maternal lineages. Based on mutations found in Samaritan paternal lineages, an estimate of a mutation rate of 0.42% (95% confidence interval of 0.22%-0.71%) across 12 Y-STRs was obtained. This estimate is slightly higher than those obtained in previous pedigree studies in other populations. The haplotypes identified in Samaritan paternal lineages that belong to the same haplogroup were used to estimate the number of generations elapsed since their most recent common ancestor (MRCA). The estimate of 80 generations corresponds with accepted traditions of the origin of this sect.

MtDNA analysis in Portuguese populations (Central Portugal and Azores Islands): polymorphic sites in control region sequences

Background: The polymorphism of the two hypervariable segments (HVI and HVII) of the control region of mtDNA was analyzed in a population of 81 unrelated individuals from Central Portugal and 48 from the Azores Islands, using a fluorescent-based electrophoresis sequencing method. Methods: Sequences have been obtained with ABI PRISM0 Big Dye Terminator and dRhodamine Terminator Cycle Sequencing Ready Reaction Kits, with Amplitaq DNA Polymerase FS, and have been detected with ABI PRISM 377 DNA sequencer. Results: In the Central Portugal population ( n=81), we observed 69 polymorphic sites of sequence in HVI region and 44 in HVII region. In the Azores population ( n=48), we observed 48 polymorphic sites of sequence in HVI region and 24 in HVII region. Conclusions: Nucleotide substitution rather than insertion/deletion (1 or 2 bp) was the majority of variation. The distribution showed a large bias towards transitional changes than transversional changes. Our sequencing results are similar to other Caucasian population data.

Analysis of mitochondrial DNA with an infrared automated DNA sequencer in a Tuscan population (Central Italy)

In our laboratory, we performed a study of the D-loop region of mtDNA in unrelated individuals, natives of Tuscany (Central Italy), using an Infrared Automated DNA Sequencer (LI-COR 4200). Taking advantage of the long read characteristics of the IR Automated Sequencer, we adopted a new strategy based on a single fragment amplification and sequencing, including the entire HVI and HVII regions. Preliminary results of the variability in the population we studied were also presented.

Genetic structure of autochthonous Basques through analysis of the HVI and HVII regions of mitochondrial DNA

Mitochondrial DNA (mtDNA) analysis has proven to be of great interest in human population studies to study phylogenetic relationships and differentiation among individuals [1]. MtDNA differs from nuclear DNA in the absence of recombination, maternal inheritance, large number of copies per cell and a mutation rate that is 5–10 times higher than that of nuclear DNA [2,3]. The non-coding region of mtDNA, known as the D-loop, accumulates more mutations than the rest of the molecule, making it a useful tool for studying short-term evolutionary phenomena [4], and the tool of choice for diagnosing matrilineal relationships. A number of scientists have studied the mtDNA of Basque populations. Bertranpetit [5] and Corte-Real [6] have analyzed the base sequences of the hypervariable segment I (HVI). All of these studies were based on an underlying assumption of homogeneous population structure in the autochthonous Basque group. The D-loop regions analyzed in this study were HVI and HVII in two autochthonous population samples Arratia and Goierri, linguistically and geographically differentiated. Since the HVII segment has not been previously studied in samples from the Basque Country, the analysis in these populations will provide better information on the variability of mtDNA in the autochthonous Basque population and its usefulness in forensic casework. Objectives: (a) to determine the homogeneity or heterogeneity of mtDNA in the autochthonous population of the Basque Country and to use the information contained in the sequence variations of the mtDNA of these fragments to analyze their phylogenetic

Mitochondrial DNA polymorphism in 50 unrelated individuals from North Italy

The hypervariable regions HVI and HVII of mitochondrial DNA (mtDNA) were sequenced in 50 unrelated individuals living in Veneto. A total of 48 different mtDNA haplotypes for HVI region (404 bp) and 49 for HVII region (313 bp) were observed. Nucleotide diversity was 0.0116 and 0.0097, respectively.

Mitochondrial DNA analysis of ancient human teeth from a XVIth century archeological excavation

Our laboratories are involved in a large-scale study investigating the different hypotheses about the founding Canary Islands populations—making use of physical–anthropological, genetic and archeological approaches. The main problems associated with ancient DNA studies are low yield, high fragmentation and contamination with exogenous DNA. In this study, we tried to evaluate the viability of our laboratory set-up to accomplish mitochondrial DNA (mtDNA) isolation and analysis from ancient samples. We present preliminary results of HVI region mtDNA sequencing of several teeth from the Monastery of Los Silos (North of Tenerife Island), an archeological excavation site dating back to the XVIth century, used as a graveyard for some time. We also make some considerations about the amplification yield by comparing two different PCR strategies.