Comparative genomic analysis of Leishmania (Viannia) peruviana and Leishmania (Viannia) braziliensis

Background

The Leishmania (Viannia) braziliensis complex is responsible for most cases of New World tegumentary leishmaniasis. This complex includes two closely related species but with different geographic distribution and disease phenotypes, L. (V.) peruviana and L. (V.) braziliensis. However, the genetic basis of these differences is not well understood and the status of L. (V.) peruviana as distinct species has been questioned by some.Here we sequenced the genomes of two L. (V.) peruviana isolates (LEM1537 and PAB-4377) using Illumina high throughput sequencing and performed comparative analyses against the L. (V.) braziliensis M2904 reference genome. Comparisons were focused on the detection of Single Nucleotide Polymorphisms (SNPs), insertions and deletions (INDELs), aneuploidy and gene copy number variations.

Results

We found 94,070 variants shared by both L. (V.) peruviana isolates (144,079 in PAB-4377 and 136,946 in LEM1537) against the L. (V.) braziliensis M2904 reference genome while only 26,853 variants separated both L. (V.) peruviana genomes.Analysis in coding sequences detected 26,750 SNPs and 1,513 indels shared by both L. (V.) peruviana isolates against L. (V.) braziliensis M2904 and revealed two L. (V.) braziliensis pseudogenes that are likely to have coding potential in L. (V.) peruviana. Chromosomal read density and allele frequency profiling showed a heterogeneous pattern of aneuploidy with an overall disomic tendency in both L. (V.) peruviana isolates, in contrast with a trisomic pattern in the L. (V.) braziliensis M2904 reference.Read depth analysis allowed us to detect more than 368 gene expansions and 14 expanded gene arrays in L. (V.) peruviana, and the likely absence of expanded amastin gene arrays.

Conclusions

The greater numbers of interspecific SNP/indel differences between L. (V.) peruviana and L. (V.) braziliensis and the presence of different gene and chromosome copy number variations support the classification of both organisms as closely related but distinct species.The extensive nucleotide polymorphisms and differences in gene and chromosome copy numbers in L. (V.) peruviana suggests the possibility that these may contribute to some of the unique features of its biology, including a lower pathology and lack of mucosal development.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1928-z) contains supplementary material, which is available to authorized users.     

Metastatic capability of Leishmania (Viannia) panamensis and Leishmania (Viannia) guyanensis in golden hamsters

The pattern and kinetics of internal dissemination and frequency of cutaneous metastatic lesions resulting from experimental infection of golden hamsters with Leishmania (Viannia) panamensis and Leishmania (Viannia) guyanensis were examined. Nineteen strains were evaluated: 16 L. (V.) panamensis isolated from patients and 3 L. (V.) guyanensis, 2 isolated from human cases and 1 WHO reference strain originating from a sandfly vector. Lymphatic dissemination occurred within 3 mo and was observed for 16 of 16 (100%) of L. (V.) panamensis and 3 of 3 (100%) of L. (V.) guyanensis. Parasites were cultured infrequently from liver and spleen: 3 of 125 (2%) L. (V.) panamensis and 1 of 22 (5%) L. (V.) guyanensis. Decreased frequency of isolation from the inoculation site and draining lymph nodes over time was accompanied by increased frequency of isolation from distant lymph nodes. Dilution of triturated tissue samples resulted in an increased efficiency of parasite culture. Both primary lesions and secondary cutaneous metastatic lesions were more severe in hamsters infected with L. (V.) guyanensis than with L. (V.) panamensis. Cutaneous metastatic lesions were produced more frequently by L. (V.) guyanensis, 24 of 46 hamsters (52%), than by L. (V.) panamensis, 28 of 252 hamsters (11%). Individual Leishmania strains displayed distinctive propensities to produce cutaneous metastases, manifested as a reproducible phenotype. Metastatic pathogenicity was independent of the inoculum dose, supporting the dissociation of infectivity and pathogenicity.     

Phylogeny and Molecular Identification of Vibrios on the Basis of Multilocus Sequence Analysis

We analyzed the usefulness of rpoA, recA, and pyrH gene sequences for the identification of vibrios. We sequenced fragments of these loci from a collection of 208 representative strains, including 192 well-documented Vibrionaceae strains and 16 presumptive Vibrio isolates associated with coral bleaching. In order to determine the intraspecies variation among the three loci, we included several representative strains per species. The phylogenetic trees constructed with the different genetic loci were roughly in agreement with former polyphasic taxonomic studies, including the 16S rRNA-based phylogeny of vibrios. The families Vibrionaceae, Photobacteriaceae, Enterovibrionaceae, and Salinivibrionaceae were all differentiated on the basis of each genetic locus. Each species clearly formed separated clusters with at least 98, 94, and 94% rpoA, recA, and pyrH gene sequence similarity, respectively. The genus Vibrio was heterogeneous and polyphyletic, with Vibrio fischeri, V. logei, and V. wodanis grouping closer to the Photobacterium genus. V. halioticoli-, V. harveyi-, V. splendidus-, and V. tubiashii-related species formed groups within the genus Vibrio. Overall, the three genetic loci were more discriminatory among species than were 16S rRNA sequences. In some cases, e.g., within the V. splendidus and V. tubiashii group, rpoA gene sequences were slightly less discriminatory than recA and pyrH sequences. In these cases, the combination of several loci will yield the most robust identification. We can conclude that strains of the same species will have at least 98, 94, and 94% rpoA, recA, and pyrH gene sequence similarity, respectively.     

Comparative zymographic analysis of metallopeptidase of Leishmania (Viannia) peruviana and Leishmania (Viannia) braziliensis isolates from Peru

American tegumentary leishmaniasis (ATL) in Peru is mainly associated with Leishmania (Viannia) peruviana and L. (V.) braziliensis. These parasites are genetically related, and their characterization as distinct species is controversial. Despite their genetic similarity, each species is associated with different clinical manifestations of ATL; L. (V.) peruviana causes only cutaneous leishmaniasis, whereas L. (V.) braziliensis can cause both cutaneous and mucocutaneous leishmaniasis. Because the primary cutaneous lesions caused by infection with these species are indistinguishable, it is necessary to develop a suitable method to differentiate them in order to prevent possible metastasis to oropharyngeal mucosa. In the present study, we investigated the proteolytic profile of L. (V.) peruviana and L. (V.) braziliensis isolates from Peru by zymographic analysis in SDS-PAGE copolymerized with gelatin. Enzymes were characterized according to their pH range of activity and sensitivity to distinct peptidase inhibitors. We observed that L. (V.) peruviana isolates displayed three proteolytic bands with molecular masses ranging from 55 to 80kDa, whereas L. (V.) braziliensis isolates showed six proteolytic activities between 55 and 130kDa. Using specific inhibitors, we determined that these proteolytic activities are due to metallopeptidases and present optimal activity between the pH range 5.5 and 10.0. Our results suggest that the expression of metallopeptidases in L. (V.) peruviana and L. (V.) braziliensis isolates from Peru is species-specific.     

Mucosal Leishmaniasis caused by Leishmania (Viannia) braziliensis and Leishmania (Viannia) guyanensis in the Brazilian Amazon

Background

Leishmania (Viannia) braziliensis is a parasite recognized as the most important etiologic agent of mucosal leishmaniasis (ML) in the New World. In Amazonia, seven different species of Leishmania, etiologic agents of human Cutaneous Leishmaniasis, have been described. Isolated cases of ML have been described for several different species of Leishmania: L. (V.) panamensis, L. (V.) guyanensis and L. (L.) amazonensis.

Methodology

Leishmania species were characterized by polymerase chain reaction (PCR) of tissues taken from mucosal biopsies of Amazonian patients who were diagnosed with ML and treated at the Tropical Medicine Foundation of Amazonas (FMTAM) in Manaus, Amazonas state, Brazil. Samples were obtained retrospectively from the pathology laboratory and prospectively from patients attending the aforementioned tertiary care unit.

Results

This study reports 46 cases of ML along with their geographical origin, 30 cases caused by L. (V.) braziliensis and 16 cases by L. (V.) guyanensis. This is the first record of ML cases in 16 different municipalities in the state of Amazonas and of simultaneous detection of both species in 4 municipalities of this state. It is also the first record of ML caused by L. (V.) guyanensis in the states of Pará, Acre, and Rondônia and cases of ML caused by L. (V.) braziliensis in the state of Rondônia.

Conclusions/Significance

L. (V.) braziliensis is the predominant species that causes ML in the Amazon region. However, contrary to previous studies, L. (V.) guyanensis is also a significant causative agent of ML within the region. The clinical and epidemiological expression of ML in the Manaus region is similar to the rest of the country, although the majority of ML cases are found south of the Amazon River.

Author Summary

Leishmaniasis is considered a neglected disease with 1.5 million new cases of cutaneous leishmaniasis (CL) occurring each year. In the Amazon region and in the Americas in general, ML is caused by Leishmania (Viannia) braziliensis, though in rare cases it has been related to other species. ML, which is associated with inadequate treatment of CL, normally manifests itself years after the occurrence of CL. Clinical features evolve slowly and most often affect the nasal cavity, in some cases causing perforation, or even destruction, of the septum. Diagnosis is made using the Montenegro skin test, serology and histopathology of the patients'' mucosal tissues, or by isolation of the parasites. PCR is the best way to identify the species of leishmaniasis and is therefore the diagnostic method of choice. This paper describes 46 cases of ML and their geographical origin, 30 cases associated with L. (V.) braziliensis and 16 with L. (V.) guyanensis. The species of leishmaniasis was identified using mucosal biopsies taken from Amazonian patients who were diagnosed and treated for ML in the tertiary care unit, in Manaus, Amazonas state, Brazil. This is the highest number of ML cases caused by L. (V.) guyanensis that has ever been reported.     

Imputation-Based Population Genetics Analysis of Plasmodium falciparum Malaria Parasites

Whole-genome sequencing technologies are being increasingly applied to Plasmodium falciparum clinical isolates to identify genetic determinants of malaria pathogenesis. However, genome-wide discovery methods, such as haplotype scans for signatures of natural selection, are hindered by missing genotypes in sequence data. Poor correlation between single nucleotide polymorphisms (SNPs) in the P. falciparum genome complicates efforts to apply established missing-genotype imputation methods that leverage off patterns of linkage disequilibrium (LD). The accuracy of state-of-the-art, LD-based imputation methods (IMPUTE, Beagle) was assessed by measuring allelic r² for 459 P. falciparum samples from malaria patients in 4 countries: Thailand, Cambodia, Gambia, and Malawi. In restricting our analysis to 86k high-quality SNPs across the populations, we found that the complete-case analysis was restricted to 21k SNPs (24.5%), despite no single SNP having more than 10% missing genotypes. The accuracy of Beagle in filling in missing genotypes was consistently high across all populations (allelic r², 0.87-0.96), but the performance of IMPUTE was mixed (allelic r², 0.34-0.99) depending on reference haplotypes and population. Positive selection analysis using Beagle-imputed haplotypes identified loci involved in resistance to chloroquine (crt) in Thailand, Cambodia, and Gambia, sulfadoxine-pyrimethamine (dhfr, dhps) in Cambodia, and artemisinin (kelch13) in Cambodia. Tajima’s D-based analysis identified genes under balancing selection that encode well-characterized vaccine candidates: apical merozoite antigen 1 (ama1) and merozoite surface protein 1 (msp1). In contrast, the complete-case analysis failed to identify any well-validated drug resistance or candidate vaccine loci, except kelch13. In a setting of low LD and modest levels of missing genotypes, using Beagle to impute P. falciparum genotypes is a viable strategy for conducting accurate large-scale population genetics and association analyses, and supporting global surveillance for drug resistance markers and candidate vaccine antigens.     

Intraspecific heterogeneity in the mini-exon gene localization of Leishmania (Viannia) panamensis and Leishmania (Viannia) guyanensis from Colombia

Intraspecific heterogeneity was demonstrated in the mini-exon gene localization from Leishmania (Viannia) panamensis and L. (Viannia) guyanensis. Different karyotypes were detected in human isolates circulating in endemic areas of Colombia. The presence of mini-exon gene sequences on chromosomes of different sizes, ranging from 370 to 800 kb in L. (V.) panamensis and from 500 to 800 kb in L. (V.) guyanensis, was observed and was neither strain nor species specific. In some cases, hybridization with 2 chromosomes in the same strain was observed. The variability of chromosomal localization of mini-exon gene sequences of these 2 species highlights the genetic variability of the Viannia subgenus and the potential utility of the mini-exon gene as a molecular epidemiologic marker.     

Leishmania (Viannia) lainsoni (Kinetoplastida: Trypanosomatidae), a divergent Leishmania of the Viannia subgenus--a mini review

Leishmania (Viannia) lainsoni is the Leishmania species that presents the most distinct biological (morphology, growth in axenic culture medium), biochemical (enzymatic electrophoresis profile), and molecular biology characteristics, when compared to other species of the Viannia subgenus. Development of promastigote forms of this parasite attached to the wall of the pyloric and hind gut regions of sand fly vectors is a solid characteristic that allows its positioning in the Viannia subgenus. However, taxonomic data from biochemical and molecular techniques on this Leishmania species are still not conclusive. It is evident the difficulty in taxonomically positioning this borderline Leishmania species. In this review we present the data accumulated since L. (Viannia) lainsoni has been described and we discuss its position in the Viannia subgenus.     

Susceptibility of spiny rats (Proechimys semispinosus) to Leishmania (Viannia) panamensis and Leishmania (Leishmania) chagasi

The role of Proechimys semispinosus as reservoir of Leishmania (Viannia) panamensis on the Colombian Pacific coast was experimentally evaluated. The susceptibility to L. chagasi also was assessed to determine the utility of this rodent as a model for studying reservoir characteristics in the laboratory. Wild-caught animals were screened for natural trypanosomatid infections, and negative individuals were inoculated intradermally (ID) in the snout or feet with 10(7) promastigotes of L. panamensis. L. chagasi was inoculated intracardially (10(7) promastigotes) or ID in the ear (10(8) promastigotes). PCR-hybridization showed that 15% of 33 spiny rats were naturally infected with L. Viannia sp. Animals experimentally infected with L. panamensis developed non-ulcerated lesions that disappeared by the 7th week post-infection (p.i.) and became more resistant upon reinfection. Infectivity to sand flies was low ((1/2)0-(1/4)8 infected/fed flies) and transient, and both culture and PCR-hybridization showed that L. panamensis was cleared by the 13th week p.i. Animals inoculated with L. chagasi became subclinically infected and were non-infective to sand flies. Transient infectivity to vectors of spiny rats infected with L. panamensis, combined with population characteristics, e.g., abundance, exploitation of degraded habitats and high reproductive rates, could make them epidemiologically suitable reservoirs.     

Experimental infection with Leishmania (Viannia) braziliensis and Leishmania (Leishmania) amazonensis in the marmoset, Callithrix penicillata (Primates:Callithricidae)

Fourteen marmosets (Callithrix penicillata) were inoculated intradermally with promastigotes and/or amastigotes of Leishmania (Viannia) braziliensis (L. (V) b.) strains MHOM/BR/83/LTB-300 MHOM/BR/85/LTB-12 MHOM/BR/81/LTB-179 and MHOM/BR/82/LTB-250. The evolution of subsequent lesions was studied for 15 to 75 weeks post-inoculation (PI). All but 3 of the L. (V) b. injected marmosets developed a cutaneous lesion at the point of inoculation after 3 to 9 weeks, characterized by the appearance of subcutaneous nodules containing parasites. Parasites were isolated by culture (Difco Blood Agar) from all 11 positive animals. The maximum size of the lesions was variable and ranged between 37 mm2 to 107 mm2. Ulceration of primary nodules became evident after 3 to 12 weeks in all infected marmosets, but was faster and larger in 5 of the 11 animals. The active lesions persisted in 9 out of 11 Callithrix until the end of the observation period, which varied from 15-75 weeks. In 3 animals spontaneous healing of their lesions (13 to 25 weeks, PI) was observed but with cryptic parasitism. In another 2 infected animals there was regression followed by reactivation of the cutaneous lesions. The appearance of smaller satellite lesions adjacent to primary ones, as well as metastatic lesions to the ear lobes, were documented in 2 animals. Promastigotes of L. (Leishmania) amazonensis (L. (L) a.) MHOM/BR/77/LTB-16 were inoculated in 1 marmoset. This animal remained chronically infected for 6 months and the lesion developed in a similar manner to L. (V) b. infected marmosets. No significant differences in clinical and parasitological behaviour were observed between promastigote or amastigote derived infections of the 2 species.(ABSTRACT TRUNCATED AT 250 WORDS)     

Recovery of Leishmania (Viannia) braziliensis from inoculated hamsters

Leishmania (Viannia) braziliensis has never been isolated from wild animals although it is apparently capable of inducing infections in man, dogs, and donkeys. An analysis of the standard hamster culture system for analyzing infectivity of Leishmania sp. was undertaken. Results indicate that for L. (V.) braziliensis, routine cultivation of aspirates taken from the inoculation sites of 1-mo-infected hamsters should be undertaken. Moreover, in at least 1 of the 3 strains examined, isolation of the parasite was only achieved after 84 days of cultivation.     

Leishmania (Viannia) braziliensis: New primers for identification using polymerase chain reaction

The objective of this study was to develop specific primers for Leishmania (Viannia) braziliensis species identification using PCR. The designed primers (LBF1 and LBR1) were evaluated for sensitivity and specificity using various L. (V.) braziliensis serodemes and various Leishmania species and also using Trypanosoma cruzi. A specific fragment of 536 bp was detected from 50 ng of DNA in a crude extract derived from L. (V.) braziliensis. The DNA fragment was not detected when DNA from other Leishmania species or from T. cruzi was used as template in the PCR. Furthermore, when tested with DNA from cutaneous leishmaniasis the designed primers and reaction gave positive results. Taking into consideration that the primers LBF1 and LBR1 could specifically identify L. (V.) braziliensis, they could be considered for use in L. (V.) braziliensis diagnosis and epidemiological studies.     

Population Structure and Evidence for Both Clonality and Recombination among Brazilian Strains of the Subgenus Leishmania (Viannia)

Background/Objectives: Parasites of the subgenus Leishmania (Viannia) cause varying clinical symptoms ranging from cutaneous leishmaniases (CL) with single or few lesions, disseminated CL (DL) with multiple lesions to disfiguring forms of mucocutaneous leishmaniasis (MCL). In this population genetics study, 37 strains of L. (V.) guyanensis, 63 of L. (V.) braziliensis, four of L. (V.) shawi, six of L. (V.) lainsoni, seven of L. (V.) naiffi, one each of L. (V.) utingensis and L. (V.) lindenbergi, and one L. (V.) lainsoni/L. naiffi hybrid from different endemic foci in Brazil were examined for variation at 15 hyper-variable microsatellite markers. Methodology/Principal findings: The multilocus microsatellite profiles obtained for the 120 strains were analysed using both model- and distance-based methods. Significant genetic diversity was observed for all L. (Viannia) strains studied. The two cluster analysis approaches identified two principal genetic groups or populations, one consisting of strains of L. (V.) guyanensis from the Amazon region and the other of strains of L. (V.) braziliensis isolated along the Atlantic coast of Brazil. A third group comprised a heterogeneous assembly of species, including other strains of L. braziliensis isolated from the north of Brazil, which were extremely polymorphic. The latter strains seemed to be more closely related to those of L. (V.) shawi, L. (V.) naiffi, and L. (V.) lainsoni, also isolated in northern Brazilian foci. The MLMT approach identified an epidemic clone consisting of 13 strains of L. braziliensis from Minas Gerais, but evidence for recombination was obtained for the populations of L. (V.) braziliensis from the Atlantic coast and for L. (V.) guyanensis. Conclusions/Significance: Different levels of recombination versus clonality seem to occur within the subgenus L. (Viannia). Though clearly departing from panmixia, sporadic, but long-term sustained recombination might explain the tremendous genetic diversity and limited population structure found for such L. (Viannia) strains.     

Analysis of the Population Structure of Anaplasma phagocytophilum Using Multilocus Sequence Typing

Anaplasma phagocytophilum is a Gram-negative obligate intracellular bacterium that replicates in neutrophils. It is transmitted via tick-bite and causes febrile disease in humans and animals. Human granulocytic anaplasmosis is regarded as an emerging infectious disease in North America, Europe and Asia. However, although increasingly detected, it is still rare in Europe. Clinically apparent A. phagocytophilum infections in animals are mainly found in horses, dogs, cats, sheep and cattle. Evidence from cross-infection experiments that A. phagocytophilum isolates of distinct host origin are not uniformly infectious for heterologous hosts has led to several approaches of molecular strain characterization. Unfortunately, the results of these studies are not always easily comparable, because different gene regions and fragment lengths were investigated. Multilocus sequence typing is a widely accepted method for molecular characterization of bacteria. We here provide for the first time a universal typing method that is easily transferable between different laboratories. We validated our approach on an unprecedented large data set of almost 400 A. phagocytophilum strains from humans and animals mostly from Europe. The typability was 74% (284/383). One major clonal complex containing 177 strains was detected. However, 54% (49/90) of the sequence types were not part of a clonal complex indicating that the population structure of A. phagocytophilum is probably semiclonal. All strains from humans, dogs and horses from Europe belonged to the same clonal complex. As canine and equine granulocytic anaplasmosis occurs frequently in Europe, human granulocytic anaplasmosis is likely to be underdiagnosed in Europe. Further, wild boars and hedgehogs may serve as reservoir hosts of the disease in humans and domestic animals in Europe, because their strains belonged to the same clonal complex. In contrast, as they were only distantly related, roe deer, voles and shrews are unlikely to harbor A. phagocytophilum strains infectious for humans, domestic or farm animals.     

Energetic metabolism of axenic promastigotes of Leishmania (Viannia) braziliensis

Leishmania spp are protozoans capable of carbohydrates degradation and as energy source they can use glucose, aminoacids or lipids from the environment. The products of the metabolic pathways such as organic acids may be used as an index of their energetic metabolic profile. Therefore, in this study a metabolic profile comparison was made between promastigotes from one reference strain (MHOM/BR/1975/M2903) and two different isolates of Leishmania (Viannia) braziliensis (MHOM/BR/2003/IMG3 and MHOM/BR/2005/RPL5). The parasites culture was performed in complete Grace’s culture media seeded in 24-well plates at 26 °C. During the growth curve performance samples were collected from the logarithmic and stationary phases of culture and therefore analyzed by high performance liquid chromatography (HPLC) and spectrophotometry assays to determine the concentrations of glucose, lactate, citrate, α-ketoglutarate, succinate, fumarate, malate, oxaloacetate and β-hydroxybutirate which are indicative of the energetic pathways. It was possible to detect an increase in the glucose from the stationary phase from the M2903 strain when compared to the logarithmic phase while in the IMG3 and RPL5 isolates there was a decrease (p < 0.05). The spectrophotometric and chromatographic results indicated that the logarithmic phase which presents higher energy consumption due to the intense replication rate have the energetic pathways intensified. It was also possible to note some metabolic differences between the analyzed parasites which may indicate possible adaptations of the parasite when facing different environmental and physiological conditions during its life cycle and that these differences may help in the understanding of the diversity of the host-parasite relationship from Leishmania parasites.     

An outbreak of human Leishmania (Viannia) braziliensis infection.

The occurrence of acute cutaneous leishmaniasis among inhabitants of 10 farms within 10 Km of the hamlet of Corte de Pedra, Bahia, Brazil was studied prospectively from 1984-1989. A mean population of 1,056 inhabitants living in 146 houses were visited every 6 months and the number of skin ulcers recorded. A leishmanin skin test survey was done people with suggestive skin scars or active disease in 1984. The incidence of skin ulcers due to Leishmania (Viannia) braziliensis (Lvb) reached 83/1,000 inhabitants but declined sharply in the subsequent 2 years. Retrospective data shows that leishmaniasis is a sporadic endemic disease. Although the reasons for this epidemic are unclear some possible aetiological factors are discussed.     

New Insights into the Phylogeny and Gene Context Analysis of Binder of Sperm Proteins (BSPs)

Seminal plasma (SP) proteins support the survival of spermatozoa acting not only at the plasma membrane but also by inhibition of capacitation, resulting in higher fertilizing ability. Among SP proteins, BSP (binder of sperm) proteins are the most studied, since they may be useful for the improvement of semen diluents, storage and subsequent fertilization results. However, an updated and detailed phylogenetic analysis of the BSP protein superfamily has not been carried out with all the sequences described in the main databases. The update view shows for the first time an equally distributed number of sequences between the three families: BSP, and their homologs 1 (BSPH1) and 2 (BSPH2). The BSP family is divided in four subfamilies, BSP1 subfamily being the predominant, followed by subfamilies BSP3, BSP5 and BSP2. BSPH proteins were found among placental mammals (Eutheria) belonging to the orders Proboscidea, Primates, Lagomorpha, Rodentia, Chiroptera, Perissodactyla and Cetartiodactyla. However, BSPH2 proteins were also found in the Scandentia order and Metatheria clade. This phylogenetic analysis, when combined with a gene context analysis, showed a completely new evolutionary scenario for the BSP superfamily of proteins with three defined different gene patterns, one for BSPs, one for BSPH1/BSPH2/ELSPBP1 and another one for BSPH1/BSPH2 without ELSPBP1. In addition, the study has permitted to define concise conserved blocks for each family (BSP, BSPH1 and BSPH2), which could be used for a more reliable assignment for the incoming sequences, for data curation of current databases, and for cloning new BSPs, as the one described in this paper, ram seminal vesicle 20 kDa protein (RSVP20, Ovis aries BSP5b).     

鹿科动物线粒体控制区序列分析与系统进化

通过测定鹿科麂亚科中的小麂、赤麂和黑麂的线粒体全基因组,从而定位它们的控制区,并从GenBank获得鹿科另外3个亚科9种动物的线粒体控制区全序列。利用MEGA软件计算了各物种控制区序列的碱基组成、遗传距离和遗传相似度,通过比较序列同源性,以羊线粒体控制区序列为外群,构建NJ分子系统树,探讨了鹿科4个亚科12种动物的系统进化关系。序列分析表明,鹿科12种动物控制区序列的碱基长度在909～1049bp之间,A T含量约占62．06％,其中363个核苷酸位点存在变异(约占34％)。系统进化关系结果表明：(1)以线粒体控制区构建的鹿科12种动物分子系统树基本与NCBI分类一致;(2)美洲鹿亚科驼鹿属驼鹿在鹿科这12种动物中处于最为原始的地位;(3)小麂比赤麂和黑麂更为原始;(4)獐亚科獐属的獐与美洲鹿亚科狍鹿属的狍鹿和美洲狍鹿聚为一支。