Multiple mutations in heterogeneous miltefosine-resistant Leishmania major population as determined by whole genome sequencing

Background

Miltefosine (MF) is the first oral compound used in the chemotherapy against leishmaniasis. Since the mechanism of action of this drug and the targets of MF in Leishmania are unclear, we generated in a step-by-step manner Leishmania major promastigote mutants highly resistant to MF. Two of the mutants were submitted to a short-read whole genome sequencing for identifying potential genes associated with MF resistance.

Methods/Principal Findings

Analysis of the genome assemblies revealed several independent point mutations in a P-type ATPase involved in phospholipid translocation. Mutations in two other proteins—pyridoxal kinase and α-adaptin like protein—were also observed in independent mutants. The role of these proteins in the MF resistance was evaluated by gene transfection and gene disruption and both the P-type ATPase and pyridoxal kinase were implicated in MF susceptibility. The study also highlighted that resistance can be highly heterogeneous at the population level with individual clones derived from this population differing both in terms of genotypes but also susceptibility phenotypes.

Conclusions/Significance

Whole genome sequencing was used to pinpoint known and new resistance markers associated with MF resistance in the protozoan parasite Leishmania. The study also demonstrated the polyclonal nature of a resistant population with individual cells with varying susceptibilities and genotypes.     

A complete Neandertal mitochondrial genome sequence determined by high-throughput sequencing

A complete mitochondrial (mt) genome sequence was reconstructed from a 38,000 year-old Neandertal individual with 8341 mtDNA sequences identified among 4.8 Gb of DNA generated from approximately 0.3 g of bone. Analysis of the assembled sequence unequivocally establishes that the Neandertal mtDNA falls outside the variation of extant human mtDNAs, and allows an estimate of the divergence date between the two mtDNA lineages of 660,000 +/- 140,000 years. Of the 13 proteins encoded in the mtDNA, subunit 2 of cytochrome c oxidase of the mitochondrial electron transport chain has experienced the largest number of amino acid substitutions in human ancestors since the separation from Neandertals. There is evidence that purifying selection in the Neandertal mtDNA was reduced compared with other primate lineages, suggesting that the effective population size of Neandertals was small.     

Thrombin-bound conformation of the C-terminal fragments of hirudin determined by transferred nuclear Overhauser effects

The interaction of the C-terminal fragments (residues 52-65 and 55-65) of the thrombin-specific inhibitor hirudin with bovine thrombin was studied by use of one- and two-dimensional NMR techniques in aqueous solution. Thrombin induces specific line broadening of the proton resonances of residues Asp(55) to Gln(65) of the synthetic hirudin fragments H-Asn-Asp-Gly-Asp(55)-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr(63)-Leu-Gln-COOH and acetyl-Asp(55)-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr(63)-Leu-Gln-COOH. This demonstrates that residues 55-65 are the predominant binding site of hirudin fragments with thrombin. Hirudin fragments take on a well-defined structure when bound to thrombin as indicated by several long-range transferred NOEs between the backbone and side-chain protons of the peptides, but they are not structured when free in solution. Particularly, transferred NOEs exist between the alpha CH proton of Glu(61) and the NH proton of Leu(64) [d alpha N(i,i+3)], between the alpha CH proton of Glu(61) and the beta CH2 protons of Leu(64) [d alpha beta(i,i+3)], and between the alpha CH proton of Glu(62) and the gamma CH2 protons of Gln(65) [d alpha gamma(i,i+3)]. These NOEs are characteristic of an alpha-helical structure involving residues Glu(61) to Gln(65). There are also NOEs between the side-chain protons of residues Phe(56), Ile(59), Pro(60), Tyr(63), and Leu(64). Distance geometry calculations suggest that in the structure of the thrombin-bound hirudin peptides all the charged residues lie on the opposite side of a hydrophobic cluster formed by the nonpolar side chains of residues Phe(56), Ile(59), Pro(60), Tyr(63), and Leu(64).     

Whole-genome sequencing of eukaryotes: From sequencing of DNA fragments to a genome assembly

Rapid advances in sequencing technologies of second- and even third-generation made the whole genome sequencing a routine procedure. However, the methods for assembling of the obtained sequences and its results require special consideration. Modern assemblers are based on heuristic algorithms, which lead to fragmented genome assembly composed of scaffolds and contigs of different lengths, the order of which along the chromosome and belonging to a particular chromosome often remain unknown. In this regard, the resulting genome sequence can only be considered as a draft assembly. The principal improvement in the quality and reliability of a draft assembly can be achieved by targeted sequencing of the genome elements of different size, e.g., chromosomes, chromosomal regions, and DNA fragments cloned in different vectors, as well as using reference genome, optical mapping, and Hi-C technology. This approach, in addition to simplifying the assembly of the genome draft, will more accurately identify numerical and structural chromosomal variations and abnormalities of the genomes of the studied species. In this review, we discuss the key technologies for the genome sequencing and the de novo assembly, as well as different approaches to improve the quality of existing drafts of genome sequences.     

Phylogenetic analysis and evolution of the genusJuglans (Juglandaceae) as determined from nuclear genome RFLPs

RFLPs were studied in 13Juglans species to determine phylogenetic relationships inJuglans. Allele frequency data were used to generate genetic distance matrices and fragment data were used to generate genetic distances based upon shared-fragments and to perform parsimony analysis. Although similar cluster analyses result from analysing allelic and shared-fragment distance, the two types of distance values displayed variable correspondence with each other. Parsimony analysis produced a tree similar to distance data trees, but with additional phylogenetic resolution agreeing with previous systematic studies. All analyses indicate an ancient origin ofJ. regia, previously considered a recently derived species.     

Construction of a cloned library of the EcoRI fragments from the human cytomegalovirus genome (strain AD169).

The DNA genome of human cytomegalovirus (HCMV) strain AD169 is 158 x 10(6) Mr. Cleavage of the HCMV DNA with the restriction endonuclease EcoRI yields 35 major fragments ranging in size from 0.54 x 10(6) Mr. We have constructed a cloned library of the EcoRI fragments of this strain of HCMV, using the plasmid pACYC184 and the recipient bacterium Escherichia coli strain HB101 RecA-. The viral origin of the cloned inserts was determined by hybridization to viral DNA. The fragments were characterized further by digestion with other restriction enzymes. Several clones were obtained which contained sequences spanning the junction between the long (L) and short (S) components of the viral DNA sequences. These clones differed in molecular weight by multiples of 0.3 x 10(6) to 0.4 x 10(6) Mr. The variability found in the clones was also reflected in the genome. Each clone containing a junction sequence hybridized to a series of bands on Southern filters of EcoRI-digested HCMV DNA. This "ladder effect" provided evidence for a region of heterogeneity within the L-S junction.     

Toward sequencing the Tetrahymena genome: exploiting the gift of nuclear dimorphism

Important scientific discoveries have utilized the unique advantages of Tetrahymena thermophila as a research organism. Recently developed molecular genetic manipulations allow full exploitation of the many scientific dividends that would result from having its genome sequenced. As a typical ciliated protozoan, Tetrahymena exhibits "nuclear dimorphism". It possesses two differentiated forms of its nuclear genome: a globally repressed, diploid germline or micronuclear genome, and a polyploid, site-specifically fragmented somatic or macronuclear genome. The macronuclear genome is, in effect, a natural, large-insert library of the micronuclear genome. This presentation describes how the gifts of nuclear dimorphism are being exploited in the experimental analysis of molecular and cell biology. Mechanisms present in humans that are either absent in other eukaryotic microbial model systems, or not as readily accessible in them as in Tetrahymena, are especially relevant. This presentation also reviews unique tools generated by nuclear dimorphism that are being used for genetically and physically mapping the Tetrahymena genome.     

Cloning of the human cytomegalovirus genome as endonuclease XbaI fragments

Restriction enzyme XbaI DNA fragments that represent 99% of the sequences from the long and short unique as well as the repeat sequences of the human cytomegalovirus (CMV) genome have been cloned into bacterial plasmid pACYC184. The viral DNA sequences associated with the recombinant plasmids were analyzed by restriction mapping and by hybridization to fragments of authentic viral DNA. The relationship of the cloned viral DNA fragments to the XbaI physical map of the viral genome is demonstrated. Even though large recombinant plasmids ranging from approx. 39 to 1.8 kb were isolated, most if not all of the viral DNA fragments were stable during propagation in Escherichia coli HB101.     

Ionizing radiation can activate the insertion of mitochondrial DNA fragments in the nuclear genome

In analytical review is considered the possibility of the insertion of mitochondrial DNA (mtDNA) fragments into the nuclear genome of cells, exposed ionizing radiation (IR). Many studies show that integration fragment mtDNA in nuclear genome, as well as its fastening as NUMT-pseudogenes, proceed at ancient periods of the evolutions not only, but also at more late periods. The number of the investigations shows that under influence endogenous reactive oxygen species, chemical agent, UV-light and IR mtDNA is damaged with greater frequency, than nucleus DNA. Furthermore, the repair systems in mitochondria are low efficiency. In irradiated by IR cells mtDNA fragments can transition from the mitochondria to the cytoplasm. The binding of mtDNA fragment to a complex with proteins provides them the protection from nuclease destroying. Possibly, at such safe condition they and are carried to nucleus. At inductions of DNA double-strand breaks (under the action of IR and activated their reparation) mtDNA fragments may be inserted to nuclear genome. Such integration of mtDNA to nuclear genome, with shaping NUMT-pseudogenes de novo, may be proceed in irradiated cells in the course of the reparations DNA double-strand breaks by the nonhomologous end-joining pathway. These insertions of mtDNA can cardinally change the structure of nuclear genomes in area of their introduction and render the essential influence upon the realization of genetic information. Available information in literature also allows to suppose that integration mtDNA in nuclear genome can proceed and at raised genomic instability observed in cells at post radiation period. It in equal extent pertains and to malignant cells with raised by instability mitochondrial and nuclear genomes. As the most efficient agent, initiating insertion fragment mtDNA in nuclear genome, is considered ionizing radiation.     

Nucleotide sequence of the EcoRI D fragment of adenovirus 2 genome

The entire nucleotide sequence of the Ad. 2 EcoRI D fragment has been determined using the Maxam and Gilbert method. This sequence of 2678 bp contains informations relative to late mRNAs ending at position 78 and for which an AATAAA sequence corresponding to their 3' ends is found at residue number 833. Position of the PVIII mRNA is determined thus allowing deduction of the probable amino acid sequence of the PVIII protein. The position and the sequence of the first leader of early 3 mRNAs is determined as well as the sequence and position of the second early leader of region 3 mRNAs, which also correspond to the "y" leader of the fiber mRNA. Following the localization of an open reading frame in which an ATG could initiate protein synthesis it can be predicted that 3a, b, c mRNAs code for the 16K early protein and the probable amino acid sequence of this protein can be deduced. The CAGTTT sequence frequently present at the 5' end of a leader or of a mRNA body as well as the GGTGAG sequence which is found at the 3' end of several leaders were used to postulate the position of various early mRNAs of region 3 and to suggest the existence of an additional splicing event during the processing of mRNAs 3a, b and c. They were also used to predict the position of the additional "x" late leaders. The imbrication of information concerning (i) the family of late mRNAs ending at position 78, (ii) the position of the "x" leader and the "y" leader and (iii) the beginning of early region 3 is also depicted.     

Mobility of elastin chains as determined by 13C nuclear magnetic resonance

Relaxation times and integrated intensities of ¹³C have been obtained from nuclear magnetic resonance spectra of elastin in unstretched calf ligamentum nuchae and indicate that about 80% of the backbone carbonyl carbons have short rotational correlation times, τ_R ～ 40 nanoseconds. τ_R is reduced by only a factor of two when the ligament is in contact with 2 m-KCNS, a strong denaturant. By contrast, the highly ordered chains of collagen in insoluble calf achilles tendon give no spectrum until denatured in 2 m-KCNS, when t_R decreases by many orders of magnitude. These results show that elastin is composed largely of highly mobile chains under physiological conditions, suggesting that configurational entropy has an important role in its elastic properties.     

Analysis of clinical ostreid herpesvirus 1 (Malacoherpesviridae) specimens by sequencing amplified fragments from three virus genome areas

Although there are a number of ostreid herpesvirus 1 (OsHV-1) variants, it is expected that the true diversity of this virus will be known only after the analysis of significantly more data. To this end, we analyzed 72 OsHV-1 "specimens" collected mainly in France over an 18-year period, from 1993 to 2010. Additional samples were also collected in Ireland, the United States, China, Japan, and New Zealand. Three virus genome regions (open reading frame 4 [ORF4], ORF35, -36, -37, and -38, and ORF42 and -43) were selected for PCR analysis and sequencing. Although ORF4 appeared to be the most polymorphic genome area, distinguishing several genogroups, ORF35, -36, -37, and -38 and ORF42 and -43 also showed variations useful in grouping subpopulations of this virus.     

Commonly conserved genetic fragments revealed by genome profiling can serve as tracers of evolution

We developed a method to produce, identify and analyze DNA fragments for the purpose of taxonomic classification. Genome profiling (GP) is a strategy that identifies genomic DNA fragments common to closely related species without prior knowledge of the DNA sequence. Random PCR, one of the key technologies of GP, is used to produce fragments and may be used even when there are mutations at the priming site. These fragments can then be distinguished based on the information of mobility and melting pattern when subjected to temperature gradient gel electrophoresis (TGGE). Corresponding fragments among several species, designated as commonly conserved genetic fragments (CCGFs), likely have the same genetic origin or correspond to the same gene. The criteria for identification of CCGFs has been defined and presented here. To assess this prediction, some of the fragments were sequenced and were confirmed to be CCGFs. We show that genome profiles bearing evolutionarily conserved CCGFs can be used to classify organisms and trace evolutionary pathways, among other profound applications.     

Cytogenetic analysis of the tomato genome by means of induced deficiencies

Cytological studies of 74 deficiencies of tomato chromosomes induced by radiation and identified by the pseudo-dominant technique reveal the loci of 35 genes on 18 of the 24 arms of the complement. These findings integrated with data obtained from various trisomic types establish centromere positions, orientation of linkage groups, and markers on all but three of the arms. The prospects of obtaining a specific kind of deficiency for a given region were found to depend on : (1) kind of radiation applied, (2) (non-random) breakage frequency in different parts of the chromosome, (3) stability of broken ends, (4) tolerance of deficiency in different parts of the genome, and (5) relative vigor of the mutant homozygote used to detect the deficiency. Aspects of the frequently observed non-homologous pairing phenomenon are presented and discussed. Marker genes whose loci are known appear to be non-randomly distributed between and within chromosomes. Chromosome exchanges as determined by genetic crossing over and cytologically observed chiasmata are likewise non-randomly distributed between and within chromosomes.Research supported in part by a grant (GM 06209) from the National Institutes of Health, U.S. Public Health Service.     

Nucleotide sequence of the EcoRI E fragment of adenovirus 2 genome.

The entire nucleotide sequence of the Ad.2 EcoRI E fragment has been determined using the Maxam and Gilbert method. This sequence of 2222 bp, which maps between coordinate 83.4 and 89.7 contains information relative to the early 3 region and to the fiber gene. Altogether with fragment EcoRI D which has been recently sequenced, they cover the entire Early 3 region in which several mRNA were mapped. The aminoacid sequence of the 16K and 14K protein is deduced. The localization of the 14.5K mRNA directing the synthesis of the third E3 known protein is discussed, as well as the hypothetical existence of three other early 3 proteins, which would have a molecular weight of 11K. The initiator ATG triplet of the fiber protein has been found at coordinate 86.1, it is followed up to the end of the fragment by an open reading frame allowing deduction of 80% of the aminoacid sequence of this protein. Sequences known to be frequently present at the border of exon sequence were used to tentatively localize the additional "Z" late leader.     

Novel multi-nucleotide polymorphisms in the human genome characterized by whole genome and exome sequencing

Genomic sequence comparisons between individuals are usually restricted to the analysis of single nucleotide polymorphisms (SNPs). While the interrogation of SNPs is efficient, they are not the only form of divergence between genomes. In this report, we expand the scope of polymorphism detection by investigating the occurrence of double nucleotide polymorphisms (DNPs) and triple nucleotide polymorphisms (TNPs), in which two or three consecutive nucleotides are altered compared to the reference sequence. We have found such DNPs and TNPs throughout two complete genomes and eight exomes. Within exons, these novel polymorphisms are over-represented amongst protein-altering variants; nearly all DNPs and TNPs result in a change in amino acid sequence and, in some cases, two adjacent amino acids are changed. DNPs and TNPs represent a potentially important new source of genetic variation which may underlie human disease and they should be included in future medical genetics studies. As a confirmation of the damaging nature of xNPs, we have identified changes in the exome of a glioblastoma cell line that are important in glioblastoma pathogenesis. We have found a TNP causing a single amino acid change in LAMC2 and a TNP causing a truncation of HUWE1.     

Sympatric genome size variation and hybridization of four oak species as determined by flow cytometry genome size variation and hybridization

The Quercus species serve as a powerful model for studying introgression in relation to species boundaries and adaptive processes. Coexistence of distant relatives, or lack of coexistence of closely relative oak species, introgression may play a role. In the current study, four closely related oak species were found in Zijinshan, China. We generated a comprehensive genome size (GS) database for 120 individuals of four species using flow cytometry‐based approaches. We examined GS variability within and among the species and hybridization events among the four species. The mean GSs of Q. acutissima, Q. variabilis, Q. fabri, and Q. serrata var. brevipetiolata were estimated to be 1.87, 1.92, 1.97, and 1.97 pg, respectively. The intraspecific and interspecific variations of GS observed among the four oak species indicated adaptation to the environment. Hybridization occurred both within and between the sections. A hybrid offspring was produced from Q. fabri and Q. variabilis, which belonged to different sections. The GS evolutionary pattern for hybrid species was expansion. Hybridization between the sections may be affected by habitat disturbance. This study increases our understanding of the evolution of GS in Quercus and will help establish guidelines for the ecological protection of oak trees.