Mariano Hernández

Mitochondrial DNA analysis of Northwest African populations reveals genetic exchanges with European, Near-Eastern, and sub-Saharan populations

Genetic studies have emphasized the contrast between North African and sub‐Saharan populations, but the particular affinities of the North African mtDNA pool to that of Europe, the Near East, and sub‐Saharan Africa have not previously been investigated. We have analysed 268 mtDNA control‐region sequences from various Northwest African populations including several Senegalese groups and compared these with the mtDNA database. We have identified a few mitochondrial motifs that are geographically specific and likely predate the distribution and diversification of modern language families in North and West Africa. A certain mtDNA motif (16172C, 16219G), previously found in Algerian Berbers at high frequency, is apparently omnipresent in Northwest Africa and may reflect regional continuity of more than 20000 years. The majority of the maternal ancestors of the Berbers must have come from Europe and the Near East since the Neolithic. The Mauritanians and West‐Saharans, in contrast, bear substantial though not dominant mtDNA affinity with sub‐Saharans.

Phylogeographic patterns of mtDNA reflecting the colonization of the Canary Islands

Although the Canary Islands were settled by humans, possibly of Berber origin, as late as 2500 years ago, the precise course and numbers of early migrations to the archipelago remain controversial. We have therefore analysed mtDNA variation (HVS‐I as well as selected RFLP sites) in 300 individuals from the seven Canary Islands. The distribution and variation across the islands in a specific mtDNA clade of Northwest African ancestry suggest that there was one dominant initial settlement process that affected all the islands, from east to west. This indicates that a certain genetic affinity of present‐day Canary Islanders to Northwest African Berbers mainly stems from the autochthonous population rather than slaves captured on the neighbouring African coast. The slave trade after the European conquest left measurable, though minor, traces in the mtDNA pool of the Canary Islands, which in its majority testifies to the European immigration.

Genetic relationship between the Canary Islanders and their African and Spanish ancestors inferred from mitochondrial DNA sequences

Nucleotide sequences of the hypervariable segment I of the control region of the mtDNA were determined in 101 individuals: 54 Canary Islanders, 18 North African Berbers, 18 Spanish mainlanders and 11 sub‐Saharan Guineans. In spite of the fact that only members of the Fang tribe were analysed, nucleotide diversity in Guineans (θ× 100 = 2·33)is one of the highest found in African populations.

Genetic diversity of bradyrhizobial populations from diverse geographic origins that nodulate Lupinus spp. and Ornithopus spp.

The genetic diversity of 45 bradyrhizobial isolates that nodulate several Lupinus and Ornithopus species in different geographic locations was investigated by 16S rDNA PCR-RFLP and sequence analysis, 16S-23S rDNA intergenic spacer (IGS) PCR-RFLP analysis, and ERIC-PCR genomic fingerprinting. Reference strains of Bradyrhizobium japonicum, B. liaoningense and B. elkanii and some Canarian isolates from endemic woody legumes in the tribe Genisteae were also included. The 16S rDNA-RFLP analysis resolved 9 genotypes of lupin isolates, a group of fourteen isolates presented restriction-genotypes identical or very similar to B. japonicum, while another two main groups of isolates (69%) presented genotypes that clearly separated them from the reference species of soybean. 16S rDNA sequencing of representative strains largely agreed with restriction analysis, except for a group of six isolates, and showed that all the lupin isolates are relatives of B. japonicum, but different lineages were observed. The 16S-23S IGS-RFLP analysis showed a high resolution level, resolving 19 distinct genotypes among 30 strains analysed, and so demonstrating the heterogeneity of the 16S-RFLP groups. ERIC-PCR fingerprint analysis showed an enormous genetic diversity producing a different pattern for each but two of the isolates. Phylogeny of nodC gene was independent from the 16S rRNA phylogeny, and showed a tight relationship in the symbiotic region of the lupin isolates with isolates from Canarian genistoid woody legumes, and in concordance, cross-nodulation was found. We conclude that Lupinus is a promiscuous host legume that is nodulated by rhizobia with very different chromosomal genotypes, which could even belong to several species of Bradyrhizobium. No correlation among genomic background, original host plant and geographic location was found, so, different chromosomal genotypes could be detected at a single site and in a same plant species, on the contrary, an identical genotype was detected in very different geographical locations and plants.

Analysis of the expression of neurokinin B, kisspeptin, and their cognate receptors NK3R and KISS1R in the human female genital tract

OBJECTIVE To investigate the presence of neurokinin B (NKB)/NK(3) receptor (NK(3)R) and kisspeptin/KISS1 receptor (KISS1R) messenger RNA (mRNA) and proteins throughout the human female genital tract. DESIGN In vitro study. SETTING Academic research laboratories and academic hospitals. PATIENT(S) Fifteen reproductive-age women and 16 postmenopausal women provided fresh samples of uterus, ovary, or oviduct, and 12 women provided archival samples of endometrium or oviduct. INTERVENTION(S) Fresh and archival samples of uterus, ovary, and oviduct obtained from reproductive-age and postmenopausal women. MAIN OUTCOME MEASURE(S) Results of reverse-transcription polymerase chain reaction (RT-PCR) and immunohistochemistry to investigate the pattern of expression of NKB/NK(3)R and kisspeptin/KISS1R in target tissues. RESULT(S) Expression of the genes encoding NKB (TAC3) and NK(3)R (TACR3), and kisspeptin (KISS1) and its receptor (KISS1R) was found in the uterus, ovary, and oviduct. Both NKB and NK(3)R immunoreactivity was detected in the endometrium, the oviduct, and the ovary, with marked expression in endometrial and oviductal epithelial cells, where intense coexpression of kisspeptin and KISS1R was also detected. Positive staining for NKB and NK(3)R was found in the myometrium where, in contrast, kisspeptin and KISS1R were absent. CONCLUSION(S) NKB/NK(3)R and kisspeptin/KISS1R are present in female peripheral reproductive tissues with colocalization of both systems in some non-neuronal cell populations of the human female genital tract. Our findings are compatible with a potential modulatory role of NKB and kisspeptin at peripheral reproductive tissues.

Mitochondrial DNA evolution in the obscura species subgroup of Drosophila.

SummaryMitochondrial DNA (mtDNA) restriction site maps for nine species of theDrosophila obscura subgroup and forDrosophila melanogaster were established. Taking into account all restriction enzymes (12) and strains (45) analyzed, a total of 105 different sites were detected, which corresponds to a sample of 3.49% of the mtDNA genome. Based on nucleotide divergences, two phylogenetic trees were constructed assuming either constant or variable rates of evolution. Both methods led to the same relationships. Five differentiated clusters were found for theobscura subgroup species, one Nearctic, represented byDrosophila pseudoobscura, and four Palearctic, two grouping the related triads of speciesDrosophila subobscura, Drosophila madeirensis, Drosophila guanche, andDrosophila ambigua, Drosophila obscura, Drosophila subsilvestris, and two more represented by one species each,Drosophila bifasciata, andDrosophila tristis. The different Palearctic clusters are as distant between themselves as with the Nearctic one. For the related speciesD. subobscura, D. madeirensis, andD. guanche, the pairD. subobscura-D. madeirensis is the closest one. The relationships found by nucleotide divergence were confirmed by differences in mitochondrial genome size, with related species sharing similar genome lengths and differing from the distant ones. The total mtDNA size range for theobscura subgroup species was from 15.5 kb forD. pseudoobscura to 17.1 forD. tristis.

Structure and evolution of the mitochondrial DNA complete control region in the Drosophila subobscura subgroup

The complete A + T‐rich region of mitochondrial DNA (mtDNA) has been cloned and sequenced in the species of the Drosophila subobscura subgroup D. subobscura, D. madeirensis and D. guanche. Comparative analysis of these sequences with others already published has identified new sequence motifs that are conserved in Drosophila and other insects. A putative bi‐directional promoter and a stop signal are proposed to be involved in the primary mtDNA strand replication of Drosophila. This region strongly resolves relationships of the species included in a phylogenetic analysis, both for closely related species and also at deeper phylogenetic levels when only the left and central domains are taken into account.

Evolution and biogeography of the genus Tarentola (Sauria: Gekkonidae) in the Canary Islands, inferred from mitochondrial DNA sequences

Sequences from fragments of the 12S ribosomal RNA and cytochrome b mitochondrial genes were used to analyze phylogenetic relationships among geckos of genus Tarentola from the Canary Islands. A surprisingly high level of within island differentiation was found in T. delalandii in Tenerife and T. boettgeri in Gran Canaria. Molecular differentiation between populations of T. angustimentalis on Lanzarote and Fuerteventura, and between Moroccan and Iberian Peninsula T. mauritanica, also indicate that at least two subspecies should be recognized within each of them. Phylogenetic relationships among these species reveals a higher level of differentiation and a more complex colonization pattern than those found for the endemic genus Gallotia. Lack of evidence for the presence of T. boettgeri bischoffi on the island of Madeira does not seem to support the origin of T. delalandii, T. gomerensis and the canarian subspecies of T. boettgeri from this island, whereas molecular data confirms that T. angustimentalis is a sister species of the continental T. mauritanica. Several independent colonization events from the continent and the extinction of some species are probably responsible for the current distribution of Tarentola in the Canary Islands.

Characterization of tachykinin receptors in the uterus of the oestrogen-primed rat

The aim of our study was to characterize the tachykinin receptor population in the oestrogen‐primed rat uterus. For this purpose, we investigated the receptor type(s) responsible for tachykinin‐induced contraction of longitudinally‐arranged smooth muscle layer. The effects of substance P (SP), neurokinin A (NKA), neurokinin B (NKB) and several of their analogues with well‐defined selectivities for tachykinin NK1, NK2 and NK3 receptors were studied and their inhibition by the selective nonpeptide tachykinin receptor antagonists (S)1‐(2‐[3‐(3,4‐dichlorophenyl)‐1‐(3‐isopropoxyphenylacetyl)piperidin‐3‐yl]ethyl)‐4‐phenyl‐1‐azoniabicyclo[2.2.2]octane chloride (SR 140333, NK1‐selective), (S)‐N‐methyl‐N[4‐(4‐acetylamino‐4‐phenylpiperidino)‐2‐(3,4‐dichlorophenyl)butyl]benzamide (SR 48968, NK2‐selective) and (R)‐(N)‐(1‐(3‐(1‐benzoyl‐3‐(3,4‐dichlorophenyl)piperidin‐3‐yl)propyl)‐ 4‐phenylpiperidin‐ 4‐yl)‐N‐ methylacetamide (SR 142801, NK3‐selective) was evaluated. Additionally, expression of tachykinin receptor mRNA was examined by using the reverse transcription‐polymerase chain reaction (RT‐PCR). SP, NKA, [Nle10]‐NKA(4‐10), the analogue with selectivity at the tachykinin NK2 receptor type, and NKB elicited concentration‐dependent contractions of the rat uterus. The pD2 values were 5.95±0.19; 6.73±0.21; 7.53±0.12 and 5.76±0.21, respectively. The selective agonist for the tachykinin NK1 receptor [Sar9Met(O2)11]‐SP produced a small phasic response in the nanomolar concentration range. The selective tachykinin NK3 receptor agonist [MePhe7]‐NKB failed to induce any significant contraction. In the presence of the neutral endopeptidase inhibitor phosphoramidon (1 μM), the log concentration‐response curves to exogenous tachykinins and their analogues were shifted significantly leftwards. The pD2 values were 6.12±0.10, 8.04±0.07, 7.89±0.03 and 6.59±0.07 for SP, NKA, [Nle10]‐NKA(4‐10) and NKB, respectively. In the presence of phosphoramidon (1 μM), [Sar9Met(O2)11]‐SP (1 nM–0.3 μM) induced concentration‐dependent contractions of increasing amplitude when only one concentration of drug was applied to each uterine strip and the pD2 value was 7.61±0.89. [MePhe7]‐NKB induced small, inconsistent contractions and, therefore, a pD2 value could not be calculated. In experiments performed in the presence of phosphoramidon (1 μM), SR 48968 (3 nM–0.1 μM) caused parallel and rightward shifts in the log concentration‐response curves of NKA. The calculated pKB value was 9.16±0.08 and the slope of the Schild regression was 1.28±0.24. SR 48968 (0.1 μM) also antagonized responses to SP with an apparent pKB value of 7.63±0.13. SR 48968 (0.1 μM) inhibited contractions elicited by NKB (1 nM–3 μM) and [Nle10]‐NKA(4–10) (0.1 nM–3 μM) but had no effect on the response evoked by [Sar9Met(O2)11]‐SP (0.1 μM). SR 140333 (0.1 μM) inhibited responses to SP with an apparent pKB value of 7.19±0.22. This compound did not significantly affect responses to NKA, [Nle10]‐NKA(4‐10) and NKB, but suppressed [Sar9Met(O2)11]‐SP (0.1 μM)‐induced contraction. SR 142801 (0.1 μM) had no effect on responses to natural tachykinins or their analogues. Total RNA was extracted from some of the uteri used in functional studies. RT‐PCR assays revealed single bands corresponding to the expected product sizes encoding cDNA for tachykinin NK1 (587 base pairs) and NK2 receptors (491 base pairs) (n=6 different animals). A very low abundance transcript corresponding to the 325 base pairs product expected for the tachykinin NK3 receptor was detected. The present data show that functionally active tachykinin NK1 and NK2 receptors are expressed in the oestrogen‐primed rat uterus. The NK2 receptor type seems to be the most important one involved in the contractile responses elicited by tachykinins. NK3 receptors are present in trace amounts and seem not to be involved in tachykinin‐induced contractions.

Mesorhizobial strains nodulating Anagyris latifolia and Lotus berthelotii in Tamadaya ravine (Tenerife, Canary Islands) are two symbiovars of the same species, Mesorhizobium tamadayense sp. nov.

Barranco de Tamadaya is a deep ravine located in southern Tenerife, which is included within a protected area where several endemic plants grow. Among them, two legumes are catalogued as critically endangered, Anagyris latifolia and Lotus berthelotii. Rhizobial strains isolated from their root nodules grown in soil samples from this ravine harboured symbiotic genes belonging to two distant symbiovars, but they shared identical 16S rRNA gene sequences (rrs). The phylogeny based on the rrs sequences placed these isolates in a separate subbranch that did not include any of the currently recognised Mesorhizobium species, but the resolution of the ribosomal tree did not permit further taxonomic conclusions. Nevertheless, multilocus sequence analysis (MLSA) of four housekeeping genes (atpD, recA, glnII and dnaK) and the rrs gene generated a highly supported Bayesian phylogeny, identifying these isolates as a new Mesorhizobium lineage. DNA-DNA hybridisation homology percentages were lower than 30% compared to type strains of the closest related species, and supported the phylogenetic data. Phenotypic characterisation also distinguished this lineage from the other closest Mesorhizobium species. The polyphasic approach thus confirmed that the isolates represented a novel species for which we propose the name Mesorhizobium tamadayense sp. nov. The type strain is Ala-3(T) (CECT 8040(T), LMG 26736(T)).