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7.

Zachary D. Stephens Skylar Y. Lee Faraz Faghri Roy H. Campbell Chengxiang Zhai Miles J. Efron Ravishankar Iyer Michael C. Schatz Saurabh Sinha Gene E. Robinson 《PLoS biology》2015,13(7)

Genomics is a Big Data science and is going to get much bigger, very soon, but it is not known whether the needs of genomics will exceed other Big Data domains. Projecting to the year 2025, we compared genomics with three other major generators of Big Data: astronomy, YouTube, and Twitter. Our estimates show that genomics is a “four-headed beast”—it is either on par with or the most demanding of the domains analyzed here in terms of data acquisition, storage, distribution, and analysis. We discuss aspects of new technologies that will need to be developed to rise up and meet the computational challenges that genomics poses for the near future. Now is the time for concerted, community-wide planning for the “genomical” challenges of the next decade.We compared genomics with three other major generators of Big Data: astronomy, YouTube, and Twitter. Astronomy has faced the challenges of Big Data for over 20 years and continues with ever-more ambitious studies of the universe. YouTube burst on the scene in 2005 and has sparked extraordinary worldwide interest in creating and sharing huge numbers of videos. Twitter, created in 2006, has become the poster child of the burgeoning movement in computational social science [6], with unprecedented opportunities for new insights by mining the enormous and ever-growing amount of textual data [7]. Particle physics also produces massive quantities of raw data, although the footprint is surprisingly limited since the vast majority of data are discarded soon after acquisition using the processing power that is coupled to the sensors [8]. Consequently, we do not include the domain in full detail here, although that model of rapid filtering and analysis will surely play an increasingly important role in genomics as the field matures.To compare these four disparate domains, we considered the four components that comprise the “life cycle” of a dataset: acquisition, storage, distribution, and analysis ( Data Phase Astronomy Twitter YouTube Genomics Acquisition 25 zetta-bytes/year0.5–15 billion tweets/year500–900 million hours/year1 zetta-bases/year Storage 1 EB/year1–17 PB/year1–2 EB/year2–40 EB/year Analysis In situ data reductionTopic and sentiment miningLimited requirementsHeterogeneous data and analysisReal-time processingMetadata analysisVariant calling, ~2 trillion central processing unit (CPU) hoursMassive volumesAll-pairs genome alignments, ~10,000 trillion CPU hours Distribution Dedicated lines from antennae to server (600 TB/s)Small units of distributionMajor component of modern user’s bandwidth (10 MB/s)Many small (10 MB/s) and fewer massive (10 TB/s) data movement Open in a separate window 相似文献

8.

One- and Two-Locus Population Models With Differential Viability Between Sexes: Parallels Between Haploid Parental Selection and Genomic Imprinting

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Alexey Yanchukov 《Genetics》2009,182(4):1117-1127

A model of genomic imprinting with complete inactivation of the imprinted allele is shown to be formally equivalent to the haploid model of parental selection. When single-locus dynamics are considered, an internal equilibrium is possible only if selection acts in the opposite directions in males and females. I study a two-locus version of the latter model, in which maternal and paternal effects are attributed to the single alleles at two different loci. A necessary condition for the allele frequency equilibria to remain on the linkage equilibrium surface is the multiplicative interaction between maternal and paternal fitness parameters. In this case the equilibrium dynamics are independent at both loci and results from the single-locus model apply. When fitness parameters are additive, analytic treatment was not possible but numerical simulations revealed that stable polymorphism characterized by association between loci is possible only in several special cases in which maternal and paternal fitness contributions are precisely balanced. As in the single-locus case, antagonistic selection in males and females is a necessary condition for the maintenance of polymorphism. I also show that the above two-locus results of the parental selection model are very sensitive to the inclusion of weak directional selection on the individual''s own genotypes.PARENTAL genetic effects refer to the influence of the mother''s and father''s genotypes on the phenotypes of their offspring, not attributable just to the transfer of genes. Examples have been documented across a wide range of areas of organism biology; see, for example, Wade (1998) and and22 in Rasanen and Kruuk (2007). Parental selection is a more formal concept used in theoretical modeling and concerns situations where the fitness of the offspring depends, besides other factors, on the genotypes of its parent(s) (generalizing from Kirkpatrick and Lande 1989).

TABLE 1

Frequencies of genotypes and fitness parameterizations in model 1

	Gametes/haploids	Frequency before selection	Fitness
Zygote	Gametes/haploids	Frequency before selection			Male	Female
(A)A	A	p_fp_m	1 − α	1 − δ
(A)a	1/2 A 1/2 a	p_f(1 − p_m)	1	1
(a)A	1/2 a 1/2 A	(1 − p_f)p_m	1 − α	1 − δ
(a)a	A	(1 − p_f)(1 − p_m)	1	1

Open in a separate windowParentheses in the first column indicate maternal genotype (parental selection model) or inactivation of the maternally derived allele (imprinting model). Whether selection occurs at the diploid (first column) or subsequent haploid (second column) stage does not change the resulting allele frequencies.

TABLE 2

Offspring genotypic proportions from different mating types, sorted among four phenotypic groups/combinations of maternal and paternal effects: model 2

		Offspring genotypes/phenotypes
Parental genotypes		Paternal (φ = 1)				Joint (φ = 4)
Male	Female	AB	Ab	aB	Ab	AB	Ab	aB	ab
AB	AB					1
	Ab
	aB
	ab	(1−r)/2	r/2	r/2	(1−r)/2
Ab	AB
	Ab		1
	aB					r/2	(1−r)/2	(1−r)/2	r/2
	ab
		Offspring genotypes/phenotypes
Parental genotypes		Maternal (φ = 2)				None (φ = 3)
Male	Female	AB	Ab	aB	Ab	AB	Ab	aB	ab
aB	AB
	Ab					r/2	(1 − r)/2	(1 − r)/2	r/2
	aB			1
	ab
ab	AB	(1 − r)/2			(1 − r)/2
	Ab
	aB
	ab								1

Open in a separate windowAnother well-known parent-of-origin phenomenon is genomic imprinting. Here, the level of expression of one of the alleles depends on which parent it is inherited from. Often it is difficult to tell apart the phenotypic patterns due to parental effects and genomic imprinting, and thus a problem arises in the process of identifying the candidate genes for such effects (). Analytic methods (; ; ) have been developed to quantify subtle differences between the two. In this article, I point out that a simple mathematical model, first suggested for genomic imprinting at a diploid locus, can be interpreted, without any formal changes, to describe parental selection on haploids.While there has been much progress in understanding the evolution of genomic imprinting (), including advances in modeling (, ), the population genetics theory of parental effects received less attention. Existing major-locus effect models of parental selection are single-locus, two-allele, and mostly concern uniparental (maternal) selection (Wright 1969; ; ; ), with only one specific case where the fitness effects of both parents interact studied by . No attempt to extend this theory into multilocus systems has yet been made. Considering a two-locus model with both parents playing a role in selection on the offspring is called for by the observation that many maternal and paternal effects aim at the different traits or different life stages of their progeny. Among birds, for example, body condition soon after hatching is largely determined by the mother, while paternally transmitted sexual display traits develop much later in life (Price 1998). Such effects are therefore unlikely to be regulated within a single locus. Sometimes the effects are on the same trait, but still attributed to different loci: expression of gene A^vy that causes the “agouti” phenotype (yellow fur coat and obesity) in mice is enhanced by maternal epigenetic modification (), while paternal mutations at the other locus, MommeD4, contribute to a reverse phenotypic pattern in the offspring (). The epigenetic state of the murine Axin^Fu allele is both maternally and paternally inherited ().Focusing selection on haploids reduces the number of genotypes that need to be taken into account, while preserving the main properties of the multilocus system. Genes with haploid expression and a potential of parental effects can be found in two major taxonomic kingdoms. A notable candidate is Spam1 in mice, which is expressed during spermogenesis and encodes a factor that enables sperm to penetrate the egg cumulus (). This gene remains a target for effectively haploid selection, because its product is not shared via cytoplasm bridges between developing spermatides. Mutations at Spam1 alter performance of the male gametes that carry it and might indirectly, perhaps by altering the timing of fertilization, affect the fitness of the zygote. The highest estimated number of mouse genes expressed in the male gametes is currently 2375 (Joseph and Kirkpatrick 2004), and one might expect some of them to have similar paternal effects. Plants go through a profound haploid stage in their life cycles, and genes involved at this stage have an inevitable effect on the fitness of the future generations. In angiosperms, seed development is known to be controlled by both maternal (; ) and paternal () effect genes, expressed, respectively, in female and male gametophytes.Under haploid selection, there can be no overdominance, and thus polymorphism is much more difficult to maintain than in diploid selection models (summarized in ). Nevertheless, differential or antagonistic selection between sexes can lead to a new class of stable internal equilibria in the diploid systems (Owen 1953; ; ; ; ), and I make use of this property in the haploid models developed below. In the experiment by Chippindale and colleagues (), ∼75% of the total fitness variation in the adult stage of Drosophila melanogaster was negatively correlated between males and females, which suggests that a substantial portion of the fruit fly expressed genome is under sexually antagonistic selection. I assume that the effect of either parent on the fitness of the individual depends on the sex of the latter, which in respect to modeling is equivalent to the assumption of differential viability between the sexes in the progeny of the same parent(s). Biological systems that satisfy the latter assumptions can be found among colonial green algae: many members of the order Volvocales are haploid except for the short zygotic stage, and during sexual reproduction, they are also dioecious and anisogametic. I return to this example in the discussion. The possibility that genes expressed in animal gametes may be under antagonistic selection between sexes has been discussed (). For example, a (hypothetical) mutation increasing the ATP production in mitochondria would be beneficial in sperm, because of the increased mobility of the latter, but neutral or detrimental in the egg, due to a higher level of oxidative damage to DNA ().My main purpose was to derive conditions for existence and stability of the internal equilibria of the model(s). I begin with a simple one-locus case, which can be analyzed explicitly, and show how these one-locus results can be extended to the case of two recombining loci with multiplicative fitness. Then, I assume an additive relation between the maternal and paternal effect parameters and study the special cases where parental effects are symmetric. 相似文献

9.

Characterization of a Thermostable Short-Chain Alcohol Dehydrogenase from the Hyperthermophilic Archaeon Thermococcus sibiricus

Tatiana N. Stekhanova Andrey V. Mardanov Ekaterina Y. Bezsudnova Vadim M. Gumerov Nikolai V. Ravin Konstantin G. Skryabin Vladimir O. Popov 《Applied and environmental microbiology》2010,76(12):4096-4098

Short-chain alcohol dehydrogenase, encoded by the gene Tsib_0319 from the hyperthermophilic archaeon Thermococcus sibiricus, was expressed in Escherichia coli, purified and characterized as an NADPH-dependent enantioselective oxidoreductase with broad substrate specificity. The enzyme exhibits extremely high thermophilicity, thermostability, and tolerance to organic solvents and salts.Alcohol dehydrogenases (ADHs; EC 1.1.1.1.) catalyze the interconversion of alcohols to their corresponding aldehydes or ketones by using different redox-mediating cofactors. NAD(P)-dependent ADHs, due to their broad substrate specificity and enantioselectivity, have attracted particular attention as catalysts in industrial processes (5). However, mesophilic ADHs are unstable at high temperatures, sensitive to organic solvents, and often lose activity during immobilization. In this relation, there is a considerable interest in ADHs from extremophilic microorganisms; among them, Archaea are of great interest. The representatives of all groups of NAD(P)-dependent ADHs have been detected in genomes of Archaea (, 12); however, only a few enzymes have been characterized, and the great majority of them belong to medium-chain (, , , , ) or long-chain iron-activated ADHs (, , ). Up to now, a single short-chain archaeal ADH from Pyrococcus furiosus (, ) and only one archaeal aldo-keto reductase also from P. furiosus () have been characterized.Thermococcus sibiricus is a hyperthermophilic anaerobic archaeon isolated from a high-temperature oil reservoir capable of growth on complex organic substrates (). The complete genome sequence of T. sibiricus has been recently determined and annotated (). Several ADHs are encoded by the T. sibiricus genome, including three short-chain ADHs (Tsib_0319, Tsib_0703, and Tsib_1998) (). In this report, we describe the cloning and expression of the Tsib_0319 gene from T. sibiricus and the purification and the biochemical characterization of its product, the thermostable short-chain ADH (TsAdh319).The Tsib_0319 gene encodes a protein with a size of 234 amino acids and the calculated molecular mass of 26.2 kDa. TsAdh319 has an 85% degree of sequence identity with short-chain ADH from P. furiosus (AdhA; PF_0074) (). Besides AdhA, close homologs of TsAdh319 were found among different bacterial ADHs, but not archaeal ADHs. The gene flanked by the XhoI and BamHI sites was PCR amplified using two primers (sense primer, 5′-GTTCTCGAGATGAAGGTTGCTGTGATAACAGGG-3′, and antisense primer, 5′-GCTGGATCCTCAGTATTCTGGTCTCTGGTAGACGG-3′) and cloned into the pET-15b vector. TsAdh319 was overexpressed, with an N-terminal His₆ tag in Escherichia coli Rosetta-gami (DE3) and purified to homogeneity by metallochelating chromatography (Hi-Trap chelating HP column; GE Healthcare) followed by gel filtration on Superdex 200 10/300 GL column (GE Healthcare) equilibrated in 50 mM Tris-HCl (pH 7.5) with 200 mM NaCl. The homogeneity and the correspondence to the calculated molecular mass of 28.7 kDa were verified by SDS-PAGE (). The molecular mass of native TsAdh319 was 56 to 60 kDa, which confirmed the dimeric structure in solution.The standard ADH activity measurement was made spectrophotometrically at the optimal pH by following either the reduction of NADP (in 50 mM Gly-NaOH buffer; pH 10.5) or the oxidation of NADPH (in 0.1 M sodium phosphate buffer; pH 7.5) at 340 nm at 60°C. The enzyme exhibited a strong preference for NADP(H) and broad substrate specificity (Table (Table1).1). The highest oxidation rates were found with pentoses d-arabinose (2.0 U mg⁻¹) and d-xylose (2.46 U mg⁻¹), and the highest reduction rates were found with dimethylglyoxal (5.9 U mg⁻¹) and pyruvaldehyde (2.2 U mg⁻¹). The enzyme did not reduce sugars which were good substrates for the oxidation reaction. The kinetic parameters of TsAdh319 determined for the preferred substrates are shown in Table Table2.2. The enantioselectivity of the enzyme was estimated by measuring the conversion rates of 2-butanol enantiomers. TsAdh319 showed an evident preference, >2-fold, for (S)-2-butanol over (RS)-2-butanol. The enzyme stereoselectivity is confirmed by the preferred oxidation of d-arabinose over l-arabinose (Table (Table1).1). The fact that TsAdh319 is metal independent was supported by the absence of a significant effect of TsAdh319 preincubation with 10 mM Me²⁺ for 30 min before measuring the activity in the presence of 1 mM Me²⁺ or EDTA (Table (Table3).3). TsAdh319 also exhibited a halophilic property, so the enzyme activity increased in the presence of NaCl and KCl and the activation was maintained even at concentration of 4 M and 3 M, respectively (Table (Table33).

TABLE 1.

Substrate specificity of TsAdh319

Substrate^a	Relative activity (%)
Oxidation reaction^b
Methanol	0
2-Methoxyethanol	0
Ethanol	36
1-Butanol	80
2-Propanol	100
(RS)-(±)-2-Butanol	86
(S)-(+)-2-Butanol	196
2-Pentanol	67
1-Phenylmethanol	180
1.3-Butanediol	91
Ethyleneglycol	0
Glycerol	16
d-Arabinose*	200
l-Arabinose*	17
d-Xylose*	246
d-Ribose*	35
d-Glucose*	146
d-Mannose*	48
d-Galactose*	0
Cellobiose*	71
Reduction reaction^c
Pyruvaldehyde	100
Dimethylglyoxal	270
Glyoxylic acid	36
Acetone	0
Cyclopentanone	0
Cyclohexanone	4
3-Methyl-2-pentanone*	13
d-Arabinose*	0
d-Xylose*	0
d-Glucose*	0
Cellobiose*	0

Open in a separate window^aSubstrates were present in 250 mM or 50 mM (*) concentrations.^bRelative rates, measured under standard conditions, were calculated by defining the activity for 2-propanol as 100%, which corresponds to 1.0 U mg⁻¹. Data are averages from triplicate experiments.^cRelative rates, measured under standard conditions, were calculated by defining the activity for pyruvaldehyde as 100%, which corresponds to 2.2 U mg⁻¹. Data are averages from triplicate experiments.

TABLE 2.

Apparent K_m and V_max values for TsAdh319

Coenzyme or substrate	Apparent K_m (mM)	V_max (U mg⁻¹)	k_cat (s⁻¹)
NADP^a	0.022 ± 0.002	0.94 ± 0.02	0.45 ± 0.01
NADPH^b	0.020 ± 0.003	3.16 ± 0.11	1.51 ± 0.05
2-Propanol	168 ± 29	1.10 ± 0.09	0.53 ± 0.04
d-Xylose	54.4 ± 7.4	1.47 ± 0.09	0.70 ± 0.04
Pyruvaldehyde	17.75 ± 3.38	4.26 ± 0.40	2.04 ± 0.19

Open in a separate window^aActivity was measured under standard conditions with 2-propanol. Data are averages from triplicate experiments.^bActivity was measured under standard conditions with pyruvaldehyde. Data are averages from triplicate experiments.

TABLE 3.

Effect of various ions and EDTA on TsAdh319^a

Compound	Concn (mM)	Relative activity (%)
None	0	100
NaCl	400	206
	600	227
	4,000	230
KCl	600	147
	2,000	200
	3,000	194
MgCl₂	10	78
CoCl₂	10	105
NiSO₄	10	100
ZnSO₄	10	79
FeSO₄	10	74
EDTA	1	100
EDTA	5	80

Open in a separate window^aThe activity was measured under standard conditions with 2-propanol; relative rates were calculated by defining the activity without salts as 100%, which corresponds to 0.9 U mg⁻¹. Data are averages from duplicate experiments.The most essential distinctions of TsAdh319 are the thermophilicity and high thermostability of the enzyme. The optimum temperature for the 2-propanol oxidation catalyzed by TsAdh319 was not achieved. The initial reaction rate of oxidation increased up to 100°C (Fig. (Fig.1).1). The Arrhenius plot is a straight line, typical of a single rate-limited thermally activated process, but there is no obvious transition point due to the temperature-dependent conformational changes of the protein molecule. The activation energy for the oxidation of 2-propanol was estimated at 84.0 ± 5.8 kJ·mol⁻¹. The thermostability of TsAdh319 was calculated from residual TsAdh319 activity after preincubation of 0.4 mg/ml enzyme solution in 50 mM Tris-HCl buffer (pH 7.5) containing 200 mM NaCl at 70, 80, 90, or 100°C. The preincubation at 70°C or 80°C for 1.5 h did not cause a decrease in the TsAdh319 activity, but provoked slight activation. The residual TsAdh319 activities began to decrease after 2 h of preincubation at 70°C or 80°C and were 10% and 15% down from the control, respectively. The determined half-life values of TsAdh319 were 2 h at 90°C and 1 h at 100°C.Open in a separate window FIG. 1.Temperature dependence of the initial rate of the 2-propanol reduction by TsAdh319. The reaction was initiated by enzyme addition to a prewarmed 2-propanol-NADP mixture. The inset shows the Arrhenius plot of the same data.Protein thermostability often correlates with such important biotechnological properties as increased solvent tolerance (2). We tested the influence of organic solvents at a high concentration (50% [vol/vol]) on TsAdh319 by using either preincubation of the enzyme at a concentration of 0.2 mg/ml with solvents for 4 h at 55°C or solvent addition into the reaction mixture to distinguish the effect of solvent on the protein stability and on the enzyme activity. TsAdh319 showed significant solvent tolerance in both cases (Table (Table4),4), and the effects of solvents could be modulated by salts, acting apparently as molecular lyoprotectants (). Furthermore, TsAdh319 maintained 57% of its activity in 25% (vol/vol) 2-propanol, which could be used as the cosubstrate in cofactor regeneration ().

TABLE 4.

Influence of various solvents on TsAdh319 activity^a

Solvent	Relative activity (%)^b	Relative activity (%)^c
Solvent	Relative activity (%)^b	Buffer without NaCl	Buffer with 600 mM NaCl
None	100	100	100
DMSO^d	98	0	40
DMFA^e	101	13	41
Methanol	98	25	9
Acetonitrile	95	0	0
Ethyl acetate	47	0*	33*
Chloroform	105	79*	81*
n-Hexane	105	60*	118*
n-Decane	36	91*	107*

Open in a separate window^aThe activity measured at the standard condition with 2-propanol as a substrate. Data are averages from triplicate experiments.^bPreincubation for 4 h at 55°C in the presence of 50% (vol/vol) of solvent prior the activity assay.^cWithout preincubation, solvent addition to the reaction mixture up to 50% (vol/vol) or using the buffer saturated by a solvent (*).^dDMSO, dimethyl sulfoxide.^eDMFA, dimethylformamide.From all the aforesaid we may suppose TsAdh319 or its improved variant to be interesting both for the investigation of structural features of protein tolerance and for biotechnological applications. 相似文献

10.

Ostrich Involvement in the Selection of H5N1 Influenza Virus Possessing Mammalian-Type Amino Acids in the PB2 Protein

Kyoko Shinya Akiko Makino Makoto Ozawa Jin Hyun Kim Yuko Sakai-Tagawa Mutsumi Ito Quynh Mai Le Yoshihiro Kawaoka 《Journal of virology》2009,83(24):13015-13018

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11.

Dominant Bacteria and Biomass in the Kuytun 51 Glacier

Shu-Rong Xiang Tian-Cui Shang Yong Chen Ze-Fan Jing Tandong Yao 《Applied and environmental microbiology》2009,75(22):7287-7290

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12.

RecA-Independent DNA Damage Induction of Mycobacterium tuberculosis ruvC Despite an Appropriately Located SOS Box

Lisa F. Dawson Joanna Dillury Elaine O. Davis 《Journal of bacteriology》2010,192(2):599-603

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13.

Isolation and Characterization of Xenorhabdus nematophila Transposon Insertion Mutants Defective in Lipase Activity against Tween

Gregory R. Richards Eugenio I. Vivas Aaron W. Andersen Delmarie Rivera-Santos Sara Gilmore Garret Suen Heidi Goodrich-Blair 《Journal of bacteriology》2009,191(16):5325-5331

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14.

DNA Repair at Telomeres: Keeping the Ends Intact

Christopher J. Webb Yun Wu Virginia A. Zakian 《Cold Spring Harbor perspectives in biology》2013,5(6)

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15.

Molecular Determinants of Adaptation of Highly Pathogenic Avian Influenza H7N7 Viruses to Efficient Replication in the Human Host

Emmie de Wit Vincent J. Munster Debby van Riel Walter E. P. Beyer Guus F. Rimmelzwaan Thijs Kuiken Albert D. M. E. Osterhaus Ron A. M. Fouchier 《Journal of virology》2010,84(3):1597-1606

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16.

Presence and Prevalence of Viruses in Local and Migratory Honeybees (Apis mellifera) in Massachusetts 总被引：1，自引：0，他引：1

Anna Welch Francis Drummond Sunil Tewari Anne Averill John P. Burand 《Applied and environmental microbiology》2009,75(24):7862-7865

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17.

Evolutionary Strata in a Small Mating-Type-Specific Region of the Smut Fungus Microbotryum violaceum

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Antonina A Votintseva Dmitry A. Filatov 《Genetics》2009,182(4):1391-1396

DNA sequence analysis and genetic mapping of loci from mating-type-specific chromosomes of the smut fungus Microbotryum violaceum demonstrated that the nonrecombining mating-type-specific region in this species comprises ∼25% (∼1 Mb) of the chromosome length. Divergence between homologous mating-type-linked genes in this region varies between 0 and 8.6%, resembling the evolutionary strata of vertebrate and plant sex chromosomes.EVOLUTION of mating types or sex-determining systems often involves the suppression of recombination around the primary sex-determining or mating-type-determining locus. In animals and plants, it is often an entire or almost entire chromosome (Y or W in male or female heterogametic species, respectively) that ceases to recombine with its homologous (X or Z) chromosome (; Charlesworth 2008). Self-incompatibility loci in plants are also thought to be located in regions of suppressed recombination (; ; ; ; ). Regardless of the phylogenetic position of a species, such nonrecombining regions are known to follow similar evolutionary trajectories. The nonrecombining region on the sex-specific chromosome expands in several steps, forming evolutionary strata—regions of different X/Y (or Z/W) divergence (; ; ; )—and genes in the nonrecombining regions gradually accumulate deleterious mutations that eventually render them dysfunctional (; Charlesworth 2008).Fungal mating-type systems are very diverse, with the number of mating types varying from two to several hundred (). Like sex chromosomes in several animals and plants, suppressed recombination has evolved in regions near fungal mating-type loci, including in Ustilago hordei (), Cryptococcus neoformans (), and Neurospora tetrasperma (). These species have two mating types, but no morphologically distinct sexes. The mating-type locus (the region of suppressed recombination) of C. neoformans is small (∼100 kb) compared with known sex chromosomes and contains only ∼20 genes that, unlike many sex chromosomes (Y or W chromosomes), show no obvious signs of genetic degeneration (; ). Judging from the divergence between the homologous genes on the two mating-type-specific chromosomes, C. neoformans started to evolve sex chromosomes a long time ago because silent divergence between the two mating types in the most ancient region exceeds 100% (). Genes in the younger mating-type-specific region are much less diverged between the two sex chromosomes, suggesting that the evolution of the sex locus in C. neoformans might have proceeded through several steps. The nonrecombining region around the mating-type locus of N. tetrasperma is much larger than in C. neoformans (at least 6.6 Mb), and silent divergence between homologous genes on the mating-type-specific chromosomes ranges from zero to 9%, demonstrating that these mating-type-specific chromosomes evolved recently ().M. violaceum, which causes anther smut disease in Silene latifolia and other species in the family Caryophyllaceae, has two mating types, A1 and A2 (reviewed by ), which are determined by the presence of mating-type-specific chromosomes (hereafter A1 and A2 chromosomes, or sex chromosomes) in the haploid stage of the life cycle (; ). The A1 and A2 chromosomes are distinguishable by size in pulsed-field electrophoresis, and it is possible to isolate individual chromosomes electrophoretically (). Random fragments of A1 and A2 chromosomes have previously been isolated from mating-type-specific bands of pulsed-field separated chromosomes of M. violaceum (). These fragments were assumed to be linked to mating type. The same method was used to isolate fragments of non-mating-type-specific chromosomes. On the basis of the analysis of their sequences, () proposed that mating-type-specific chromosomes in M. violaceum might be degenerate because they contained a lower proportion of protein-coding genes than other chromosomes. However, it was not determined whether the sequences isolated from the mating-type chromosomes originated from the mating-type-specific or from the recombining regions (), and the relative sizes of these regions are not known for these M. violaceum chromosomes. We tested the mating-type specificity of 86 of these fragments and demonstrate that fewer than a quarter of these loci are located in the mating-type-specific region, suggesting that the nonrecombining region on the A1 and A2 chromosomes is quite small, while the rest of the chromosome probably recombines (like pseudoautosomal regions of sex chromosomes) and is therefore not expected to undergo genetic degeneration. Genetic mapping confirms the presence of two pseudoautosomal regions in the M. violaceum mating-type-specific chromosomes.As these chromosomes are mating type specific in the haploid stage of M. violaceum, mating-type-specific loci (or DNA fragments) can be identified by testing whether they are present exclusively in A1 or A2 haploid strains. We therefore prepared haploid A1 and A2 M. violaceum cultures from S. latifolia plants from two geographically remote locations (accessions Sl405 from Sweden and Sl127 from the French Pyrenees). Haploid sporidial cultures were isolated by a standard dilution method (Kaltz and Shykoff 1997; Oudemans and Alexander 1998). Mating types were determined by PCR amplification of each culture with primers designed for A1 and A2 pheromone receptor genes linked to A1 and A2 mating types (). The primers were as follows: 5′-TGGCATCCCTCAATGTTTCC-3′ and 5′-CACCTTTTGATGAGAGGCCG-3′ for the A1 pheromone receptor (GenBank accession no. EF584742) and 5′-TGACGAGAGCATTCCTACCG-3′ and 5′-GAAGCGGAACTTGCCTTTCT-3′ for the A2 pheromone receptor (GenBank accession no. EF584741). Cultures with PCR product amplified only from an A1 or A2 pheromone receptor gene were selected for further use. The mating types of the cultures were verified by conjugating them in all combinations.The GenBank nucleotide database was searched using BLAST for sequences similar to those isolated by . Sequences with similarity to transposable elements (TE) and other repeats were excluded. The resulting set of nonredundant sequences was used to design PCR primers for 98 fragments. Half of these were originally isolated from the A1 and half from the A2 chromosomes and are hereafter called A1-NNN or A2-NNN (where NNN is the locus number; supporting information, Table S1), which does not imply that these loci are A1 or A2 specific, but merely indicates that they were originally isolated from the A1 or A2 chromosomes. Amplification of these regions from new A1 and A2 M. violaceum cultures, independently isolated by ourselves, revealed that only 5 of the 49 loci isolated from the A1 chromosome are indeed A1 specific and only 6 of 49 isolated from the A2 chromosome are A2 specific. All other loci amplified from both A1 and A2 cultures. Figure 1 illustrates some of these results from the Swedish sample (Sl405).Open in a separate window Figure 1.—Testing of mating-type specificity for loci isolated from A1 and A2 chromosomes. (a) PCR amplifications from haploid cultures from Sl405 using primers designed from six A1-originated loci. Loci in which a PCR product could be amplified only from A1 cultures (boxed) were classified as specific to mating type A1. (b) PCR tests of six A2-originated loci on the same set of haploids as in a. Loci in which a PCR product amplified only from A2 cultures (boxed) were classified as specific to mating type A2. Loci amplified from both A1 and A2 cultures are not mating type specific.The fragments that amplified from both A1 and A2 mating types may be in recombining regions, or they could be present in mating-type-specific regions on both A1 and A2 chromosomes. If they are in recombining regions, the A1- and A2-linked homologs should not be diverged from each other, but if they are in nonrecombining, mating-type-specific regions, the divergence of the A1- and A2-linked homologs should be roughly proportional to the time since recombination stopped in the region. We therefore sequenced and compared PCR fragments amplified from the two mating types of Sl405 or Sl127 cultures (GenBank accession nos. FI855822–FI856001). Sequencing of PCR products showed that 12 (4 A1 and 8 A2) loci have more than one copy, and they were excluded from further analysis. Sequences of 61 loci were identical between the A1 and A2 strains, and four loci demonstrated low total divergence (0.24–0.61%) between the two mating types (otintseva and D. Filatov, unpublished results). Thus, these loci might be located in the recombining part of the mating-type-specific chromosomes. Ten of 75 loci that amplified in both mating types demonstrated multiple polymorphisms fixed between the mating types rather than between the locations. Given that the strains that we used in the analysis originated from two geographically distant locations, it is highly unlikely that multiple polymorphisms distinguishing the A1 and A2 sequences arose purely by chance; thus, these loci are probably located in the nonrecombining mating-type-specific region of the M. violaceum A1 and A2 chromosomes.

TABLE 1

Loci from mating-type-specific chromosomes of M. violaceum used for PCR analysis and genetic map construction

		With nonzero A1/A2 divergence^{^b}
Loci	Mating type specific	<1%	>1%	With zero A1/A2 divergence^{^b}	Total
A1^{^a}	5	2 (1)	3 (3)	35 (3)	45 (7)
A2^{^a}	6	2 (0)	7 (7)	26 (3)	41 (10)
Subtotal		4 (1)	10 (10)
Total	11	14 (11)		61 (6)	86 (17)

Open in a separate window^aA1, loci originated from the A1 sex chromosome; A2, loci originated from the A2 sex chromosome.^bThe number of loci used for genetic map construction is in parentheses.To confirm the mating-type-specific or pseudoautosomal locations of the loci with and without A1/A2 divergence, we conducted genetic mapping in a family of 99 individuals, 50 of which were of mating type A1 and 49 of mating type A2. The family was generated by a cross between A1 and A2 M. violaceum strains from S. latifolia accessions Sl405 (Sweden) and Sl127 (France), respectively. The choice of strains from geographically distant locations was motivated by the hope of maximizing the number of DNA sequence differences between them that can be used as molecular genetic markers in segregation analysis. We inoculated S. latifolia seedlings with sporidial cultures of both mating types. For inoculation, petri dishes with 12-day-old seedlings of S. latifolia were flooded with 2.5 ml of inoculum suspension. Inoculum suspension consisted of equal volumes of the A1 and A2 sporidial cultures that were mixed and conjugated overnight at 14° under rotation (Biere and Honders 1996; ). Seedlings were potted 3 days after inoculation. Two months later, teliospores were collected from the flowers of the infected plant and grown in petri dishes on 3.6% potato dextrose agar medium. Haploid sporidia formed after meiosis were isolated and grown as separate cultures for DNA extraction. The mating types of single sporidia cultures were identified as described above. The loci analyzed in the segregation analysis were sequenced in the two parental haploid strains and in 99 (50 A1 and 49 A2) haploid strains that were generated in the cross. Single nucleotide differences between the parental strains were used as molecular genetic markers for segregation analysis in the progeny. The genetic map was constructed using MAPMAKER/EXP v3.0 (Lincoln et al. 1992) and MapDisto v1.7 (http://mapdisto.free.fr/).The resulting genetic map is shown in Figure 2. As expected, no recombination was observed between the 10 loci with diverged A1- and A2-linked copies. In addition, one marker with no A1/A2 divergence, A2-397, was also completely linked to the loci with significant A1/A2 divergence. This locus either may be very tightly linked to the nonrecombining mating-type-specific region or may have been added to that region more recently than the loci that had already accumulated some divergence between the alleles in the two mating types. The mating-type-specific pheromone receptor locus () and 11 mating-type-specific loci are also located in this nonrecombining region (Figure 2). Interestingly, the cluster of nonrecombining markers is flanked on both sides with markers that recombine in meiosis, demonstrating that there are pseudoautosomal regions on both ends of the mating-type-specific chromosomes.Open in a separate window Figure 2.—Genetic map of the mating-type-determining chromosome in M. violaceum. Genetic distance (in centimorgans) and the relative positions of the markers are shown to the left and the right of the chromosome, respectively. The position of the nonrecombining region corresponds to the cluster of linked markers shown on the right of the figure. Total A1/A2 divergence is shown in parentheses. Eleven mating-type-specific markers (for which sequences are available from only one mating type), located in the nonrecombining mating-type-specific region, are not shown.Our results demonstrate that although the loci reported by were isolated from the A1 and A2 chromosomes, most of these loci are not located in the nonrecombining mating-type-specific regions. In fact, the nonrecombining region might be relatively small: of 86 tested fragments, only 21 appeared to be either mating type specific or linked to the mating-type locus. Assuming that these loci represent a random set of DNA fragments isolated from the A1 and A2 chromosomes, it is possible to estimate the size of the nonrecombining region using the binomial distribution: the nonrecombining region is expected to be 24.4% (95% CI: 16.7–33.6%) of the chromosome length. As the sizes of the A1 and A2 chromosomes are ∼3.4 and 4.2 Mb long (; ), the nonrecombining region might be ∼1 Mb long.Interestingly, total A1/A2 divergence for the 11 loci with A1- and A2-linked copies mapped to the nonrecombining region varied from 0% to 8.6% (Figure 2). In addition, 11 loci amplified from only one mating type. These genes could represent degenerated genes, some of which degenerated in A1 strains, and some in A2 strains. Alternatively, they might be highly diverged genes, such that the PCR primers amplify only one allele, and not the other. Variation in divergence may be the result of the stepwise cessation of recombination between the A1 and A2 chromosomes in M. violaceum, resembling the evolutionary strata reported for human, chicken, and white campion sex chromosomes (; ; ). However, only the differences between the most and the least diverged loci are statistically significant (Devier et al. 2009), the M. violaceum mating-type region has at least three strata: one oldest stratum, including the pheromone receptor locus; a younger stratum with ∼5–9% A1/A2 divergence; and the youngest stratum with 1–4% divergence between the two mating types. There may also be an additional very recently evolved stratum containing the locus named A2-397, which is also present in all A1 strains tested, with no fixed differences between the A1 and A2 strains (No. of sites analyzedWithin A1

Within A2

Fixed differences between A1 and A2A1/A2 divergence (%)Loci^{^a}S^{^b} totalSπ (%)^{^c}Sπ (%)^{^c}A1/A2 divergence <1%A1-23645630020.4410.44A1-0456544000040.61A2-568413220.4820.4800.24A2-411480210.210010.31A1/A2 divergence >1%A1-2176679000091.35A1-12856990010.1881.49A1-199618130010.16122.02A2-4223449000092.62A2-516470140000142.98A2-404508200030.59173.64A2-4355062220.3920.39183.95A2-4734572310.2210.22214.81A2-4573031710.3300165.54A2-5755034750.9930.59398.55 Open in a separate window^aA1, loci originated from the A1 sex chromosome; A2, loci originated from the A2 sex chromosome.^bS, number of polymorphic sites.^cπ (%), average number of differences per 100 nucleotides.

TABLE 3

P-values for the 2 × 2 G-tests for significance of differences in A1/A2 divergence between the loci in the nonrecombining region

L^{^a}	S^{^b}	Locus	A2-397	A1-217	A1-128	A1-199	A2-422	A2-516	A2-404	A2-435	A2-473	A2-457
519	0	A2-397
667	9	A1-217	0.006
569	8	A1-128	0.006	0.93
618	12	A1-199	0.0007	0.41	0.48
344	9	A2-422	0.0003	0.17	0.21	0.51
470	14	A2-516	0.00003	0.06	0.086	0.28	0.76
508	17	A2-404	0	0.025	0.038	0.15	0.55	0.75
506	18	A2-435	0	0.015	0.024	0.104	0.45	0.62	0.86
457	21	A2-473	0	0.001	0.003	0.0163	0.15	0.21	0.34	0.43
303	16	A2-457	0	0.0009	0.0017	0.0097	0.09	0.13	0.203	0.26	0.69
503	39	A2-575	0	0	0	0.00001	0.002	0.002	0.003	0.006	0.055	0.199

Open in a separate windowP-values <0.05 are in boldface type.^aL, the length of the region compared.^bS, the number of nucleotide differences observed.As most of the loci isolated from the A1 and A2 chromosomes recombine in meiosis, they are not expected to degenerate. Thus, the observation of a higher proportion of TEs in these loci, compared to other chromosomes (), is unlikely to reflect genetic degeneration attributable to a lack of recombination in these loci. A higher abundance of TEs in the sequences isolated from the A1 and A2 chromosomes, as reported by , may simply reflect variation in the TE density across the genome. Thus, it remains to be seen whether M. violaceum mating-type-specific regions degenerate, similar to vertebrate Y (or W) chromosomes, or remain largely intact, as in C. neoformans (). If the latter were the case, it may suggest that nonrecombining regions in fungi do not necessarily follow the same degenerative path as animal Y and W chromosomes. The analysis of sequences from the M. violaceum genome (and perhaps other fungal genomes) will hopefully provide the answer to this question.The lack of degeneration of mating-type-specific regions in C. neoformans may be due to the relatively small size of the nonrecombining regions. The 20 genes present in this region may not be sufficient for the operation of such detrimental population genetic processes as background selection or Muller''s ratchet because the speed of these processes depends critically on the number of active genes linked together (Charlesworth 2008). Larger mating-type-specific regions in M. violaceum might contain more genes; thus, more active genetic degeneration may be expected in this species. Indeed, many strains of M. violaceum show haplolethality linked to one of the mating types (; Thomas et al. 2003; ), which may reflect the accumulation of deleterious mutations in the nonrecombining regions around the mating-type loci. Mating-type specificity of the markers that amplified in only A1 or A2 strains in this study may also reflect genetic degeneration.Another factor that may potentially prevent degeneration of genes linked to mating-type loci in fungi is the haploid expression of genes in these regions. In animals, many Y-linked genes have functional homologs on the X chromosome, and loss of the Y-linked gene may be compensated for by expression of the X-linked homologs. The haploid stage in an animal''s life cycle is very short, and very few genes are actively expressed in animal gametes (). In plants, on the other hand, a significant proportion of the genome is expressed in pollen (), and so the loss of Y-linked genes expressed in gametes may be more detrimental than in animals. Indeed, most genes isolated from the white campion X chromosome have intact Y-linked copies (; ), but due to the small number of genes available, it is still unclear whether genetic degeneration of Y-linked genes is indeed slower in this species (and in plants generally) compared to animal Y chromosomes. Haploid expression could be an even more powerful force in fungi and other organisms with haploid sexes, such as bryophytes, as most genes are expressed in the haploid stage. Further analysis of genetic degeneration in nonrecombining sex- or mating-type-specific regions in fungi and bryophytes will help to shed light on this question. 相似文献

18.

Identification of Enhancer Binding Proteins Important for Myxococcus xanthus Development

Krista M. Giglio Jessica Eisenstatt Anthony G. Garza 《Journal of bacteriology》2010,192(1):360-364

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19.

In Vitro and In Vivo Oncogenic Potential of Bovine Leukemia Virus G4 Protein

Pierre Kerkhofs Hubertine Heremans Arsène Burny Richard Kettmann Luc Willems 《Journal of virology》1998,72(3):2554-2559

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20.

Wide Variation in the Multiplicity of HIV-1 Infection among Injection Drug Users

Katharine J. Bar Hui Li Annie Chamberland Cecile Tremblay Jean Pierre Routy Truman Grayson Chuanxi Sun Shuyi Wang Gerald H. Learn Charity J. Morgan Joseph E. Schumacher Barton F. Haynes Brandon F. Keele Beatrice H. Hahn George M. Shaw 《Journal of virology》2010,84(12):6241-6247

Recent studies indicate that sexual transmission of human immunodeficiency virus type 1 (HIV-1) generally results from productive infection by only one virus, a finding attributable to the mucosal barrier. Surprisingly, a recent study of injection drug users (IDUs) from St. Petersburg, Russia, also found most subjects to be acutely infected by a single virus. Here, we show by single-genome amplification and sequencing in a different IDU cohort that 60% of IDU subjects were infected by more than one virus, including one subject who was acutely infected by at least 16 viruses. Multivariant transmission was more common in IDUs than in heterosexuals (60% versus 19%; odds ratio, 6.14; 95% confidence interval [CI], 1.37 to 31.27; P = 0.008). These findings highlight the diversity in HIV-1 infection risks among different IDU cohorts and the challenges faced by vaccines in protecting against this mode of infection.Elucidation of virus-host interactions during and immediately following the transmission event is one of the great challenges and opportunities in human immunodeficiency virus (HIV)/AIDS prevention research (-, , , ). Recent innovations involving single-genome amplification (SGA), direct amplicon sequencing, and phylogenetic inference based on a model of random virus evolution (-, ) have allowed for the identification of transmitted/founder viruses that actually cross from donor to recipient, leading to productive HIV type 1 (HIV-1) infection. Our laboratory and others have made the surprising finding that HIV-1 transmission results from productive infection by a single transmitted/founder virus (or virally infected cell) in ∼80% of HIV-infected heterosexuals and in ∼60% of HIV-infected men who have sex with men (MSM) (, , , ). These studies thus provided a precise quantitative estimate for the long-recognized genetic bottleneck in HIV-1 transmission (, -, , , , , , , , -) and a plausible explanation for the low acquisition rate per coital act and for graded infection risks associated with different exposure routes and behaviors (, ).In contrast to sexual transmission of HIV-1, virus transmission resulting from injection drug use has received relatively little attention (, , , ) despite the fact that injection drug use-associated transmission accounts for as many as 10% of new infections globally (, 46). We hypothesized that SGA strategies developed for identifying transmitted/founder viruses following mucosal acquisition are applicable to deciphering transmission events following intravenous inoculation and that, due to the absence of a mucosal barrier, injection drug users (IDUs) exhibit a higher frequency of multiple-variant transmission and a wider range in numbers of transmitted viruses than do acutely infected heterosexual subjects. We obtained evidence in support of these hypotheses from the simian immunodeficiency virus (SIV)-Indian rhesus macaque infection model, where we showed that discrete low-diversity viral lineages emanating from single or multiple transmitted/founder viruses could be identified following intravenous inoculation and that the rectal mucosal barrier to infection was 2,000- to 20,000-fold greater than with intravenous inoculation (). However, we also recognized potentially important differences between virus transmission in Indian rhesus macaques and virus transmission in humans that could complicate an IDU acquisition study. For example, in the SIV macaque model, the virus inocula can be well characterized genetically and the route and timing of virus exposure in relation to plasma sampling precisely defined, whereas in IDUs, the virus inoculum is generally undefined and the timing of virus infection only approximated based on clinical history and seroconversion testing (). In addition, IDUs may have additional routes of potential virus acquisition due to concomitant sexual activity. Finally, there is a paucity of IDU cohorts for whom incident infection is monitored sufficiently frequently and clinical samples are collected often enough to allow for the identification and enumeration of transmitted/founder viruses. To address these special challenges, we proposed a pilot study of 10 IDU subjects designed to determine with 95% confidence if the proportion of multivariant transmissions in IDUs was more than 2-fold greater than the 20% frequency established for heterosexual transmission (, , , ). A secondary objective of the study was to determine whether the range in numbers of transmitted/founder viruses in IDUs exceeded the 1-to-6 range observed in heterosexuals (, , , ). To ensure comparability among the studies, we employed SGA-direct amplicon sequencing approaches, statistical methods, and power calculations identical to those that we had used previously to enumerate transmitted/founder viruses in heterosexual and MSM cohorts (, , , , ).We first surveyed investigators representing acute-infection cohorts in the United States, Canada, Russia, and China; only one cohort—the Montreal Primary HIV Infection Cohort ()—had IDU clinical samples and clinical data available for study. The Montreal cohort of subjects with acute and early-stage HIV-1 infection was established in 1996 and recruits subjects from both academic and private medical centers throughout the city. Injection drug use is an important contributing factor to Montreal''s HIV burden, with IDUs comprising approximately 20% of the city''s AIDS cases and 35% of the cohort (21, , ). A large proportion of Montreal''s IDUs use injection cocaine, with 50 to 69% of subjects reporting cocaine as their injection drug of choice (, , , , ).Subjects with documented serological evidence of recent HIV-1 infection and a concurrent history of injection drug use were selected for study. These individuals had few or no reported risk factors for sexual HIV-1 acquisition. Clinical history and laboratory tests of HIV-1 viremia and antibody seroconversion were used to determine the Fiebig clinical stage () and to estimate the date of infection (Table (Table1).1). One subject was determined to be in Fiebig stage III, one subject was in Fiebig stage IV, five subjects were in Fiebig stage V, and three subjects were in Fiebig stage VI. We performed SGA-direct amplicon sequencing on stored plasma samples and obtained a total of 391 3′ half-genomes (median, 25 per subject; range, 19 to 167). Nine of these sequences contained large deletions or were G-to-A hypermutated and were excluded from subsequent analysis. Sequences were aligned, visually inspected using the Highlighter tool (www.hiv.lanl.gov/content/sequence/HIGHLIGHT/highlighter.html), and analyzed by neighbor-joining (NJ) phylogenetic-tree construction. A composite NJ tree of full-length gp160 env sequences from all 10 subjects (Fig. (Fig.1A)1A) revealed distinct patient-specific monophyletic lineages, each with high bootstrap support and separated from the others by a mean genetic distance of 10.79% (median, 11.29%; range, 3.00 to 13.42%). Maximum within-patient env gene diversity ranged from 0.23% to 3.34% (Table (Table1).1). Four subjects displayed distinctly lower within-patient maximum env diversities (0.23 to 0.49%) than the other six subjects (1.48% to 3.34%). The lower maximum env diversities in the former group are consistent with infection either by a single virus or by multiple closely related viruses, while the higher diversities can be explained only by transmission of more than one virus based on empirical observations (, , , ) and mathematical modeling (, ).Open in a separate window FIG. 1.NJ trees and Highlighter plots of HIV-1 gp160 env sequences. (A) Composite tree of 382 gp160 env sequences from all study subjects. The numerals at the nodes indicate bootstrap values for which statistical support exceeded 70%. (B) Subject ACT54869022 sequences suggest productive infection by a single virus (V1). (C) Subject HDNDRPI032 sequences suggest productive infection by as many as three viruses. (D) Subject HDNDRPI001 sequences suggest productive infection by at least five viruses with extensive interlineage recombination. Sequences are color coded to indicate viral progeny from distinct transmitted/founder viruses. Recombinant virus sequences are depicted in black. Methods for SGA, sequencing, model analysis, Highlighter plotting, and identification of transmitted/founder virus lineages are described elsewhere (, , , ). The horizontal scale bars represent genetic distance. nt, nucleotide.

TABLE 1.

Subject demographics and HIV-1 envelope analysis results

Subject identifier	Age (yr)	Sex^a	Fiebig stage	Estimated no. of days postinfection^b	CD4 count	Plasma viral load (log)	No. of SGA amplicons	Diversity of env genes (%)^c			No. of transmitted/ founder viruses
Subject identifier	Age (yr)	Sex^a	Fiebig stage	Estimated no. of days postinfection^b	CD4 count	Plasma viral load (log)	No. of SGA amplicons	Mean	Interquartile range	Maximum^d	Model prediction^e	Phylogenetic estimate^f
HDNDRPI034	47	M	III	29	240	7.88	163	1.07	0.55	3.34	>1	16
HDNDRPI029	18	F	IV	48	440	4.34	29	0.16	0.15	0.49	1	1
HTM385	24	M	V	62	406	5.37	22	0.12	0.08	0.27	1	1
CQLDR03	42	M	V	66	ND^g	5.01	21	0.08	0.08	0.23	1	1
HDNDRPI001	36	M	V	28	690	5.94	25	0.90	0.63	1.91	>1	5
HTM319	39	M	V	68	520	4.43	25	0.77	0.46	1.54	>1	3
HDNDRPI032	37	M	V	73	1,040	3.53	19	1.48	2.99	3.34	>1	3
ACTDM580208	39	M	VI	93	387	4.53	30	1.17	0.97	2.64	>1	3
ACT54869022	28	M	VI	68	723	3.43	27	0.07	0.04	0.24	1	1
PSL024	46	M	VI	82	340	4.46	21	0.82	0.63	1.57	>1	3

Open in a separate window^aM, male; F, female.^bNumbers of days postinfection were estimated on the basis of serological markers, clinical symptoms, or a history of a high-risk behavior leading to virus exposure.^cDiversity measurements determined by PAUP* analysis.^dThe model prediction of the maximum achievable env diversity 100 days after transmission is 0.60% (95% CI, 0.54 to 0.68%). Diversity values exceeding this range imply transmission and productive infection by more than one virus. Diversity values less than 0.54% can be explained by transmission of one virus or of multiple closely related viruses ().^eModel described in Keele et al. ().^fMinimum estimate of transmitted/founder viruses.^gND, not determined.An example of productive clinical infection by a single virus is shown in phylogenetic tree and Highlighter plots from subject ACT54869022 (Fig. (Fig.1B).1B). A similar phylogenetic pattern of single-variant transmission was found in 4 of 10 IDU subjects (Table (Table1).1). Examples of multivariant transmission are shown for subject HDNDRPI032, for whom there was evidence of infection by 3 transmitted/founder viruses (Fig. (Fig.1C)1C) and for subject HDNDRPI001, for whom there was evidence of infection by at least 5 transmitted/founder viruses (Fig. (Fig.1D).1D). One IDU subject, HDNDRPI034, had evidence of multivariant transmission to an extent not previously seen in any of 225 subjects who acquired their infection by mucosal routes (, , , ) or in any of 13 IDUs, as recently reported by Masharsky and colleagues (). We greatly extended the depth of our analysis in this subject to include 163 3′ half-genome sequences in order to increase the sensitivity of detection of low-frequency viral variants. Power calculations indicated that a sample size of 163 sequences gave us a >95% probability of sampling minor variants comprising as little as 2% of the virus population. By this approach, we found evidence of productive infection by at least 16 genetically distinct viruses (Fig. (Fig.2).2). Fourteen of these could be identified unambiguously based on the presence of discrete low-diversity viral lineages, each consisting of between 2 and 48 sequences. Two additional unique viral sequences with long branch lengths (3F8 and G10) exhibited diversity that was sufficiently great to indicate a distinct transmission event as opposed to divergence from other transmitted/founder lineages (see the legend to Fig. Fig.2).2). It is possible that still other unique sequences from this subject also represented transmitted/founder viruses, but we could not demonstrate this formally. We also could not determine if all 16 (or more) transmission events resulted from a single intravenous inoculation or from a series of inoculations separated by hours or days; however, it is likely that all transmitted viruses in this subject resulted from exposure to plasma from a single infected individual, since the maximum env diversity was only 3.34% (Fig. (Fig.1A).1A). It is also likely that transmission occurred within a brief window of time, since the period from transmission to the end of Fiebig stage III is typically only about 25 days (95% CI, 22 to 37 days) (, ) and the diversity observed in all transmitted/founder viral lineages in subject HDNDRPI034 was exceedingly low, consistent with model predictions for subjects with very recent infections (, ).Open in a separate window FIG. 2.NJ tree and Highlighter plot of HIV-1 3′ half-genome sequences from subject HDNDRPI034. Sequences emanating from 16 transmitted/founder viruses are color coded. Fourteen transmitted/founder viral lineages comprised of 2 or more identical or nearly identical sequences could be readily distinguished from recombinant sequences (depicted in black), which invariably appeared as unique sequences containing interspersed segments shared with other transmitted/founder virus lineages. The two sequences with the longest branch lengths (3F8 and G10) were interpreted to represent rare progeny of discrete transmitted/founder viruses because their unique polymorphisms far exceeded the maximum diversity estimated to occur in the first 30 days of infection (0.22%; CI, 0.15 to 0.31%) () and far exceeded the diversity observed within the other transmitted/founder virus lineages. The horizontal scale bar represents genetic distance.Lastly, we compared the multiplicity of HIV-1 transmission in the Montreal IDU subjects with that of non-IDU subjects for whom identical SGA methods had been employed. In this combined-cohort analysis, we found the frequency of multiple-variant transmission in heterosexuals to be 19% (34 of 175) and in MSM 38% (19 of 50) (Table (Table2)2) (). The current study was powered to detect a >2-fold difference in multivariant transmission between IDUs and heterosexual subjects; in fact, we observed a 3-fold-higher frequency of multiple-variant transmission in Montreal IDUs (6 of 10 subjects [60%]) than in heterosexuals (odds ratio, 6.14; 95% CI, 1.37 to 31.27; Fisher exact test, P = 0.008) and a 1.5-fold-higher frequency in Montreal IDUs than in MSM (odds ratio, 2.41; 95% CI, 0.50 to 13.20; P = 0.294, not significant). In addition, we found that the range of numbers of transmitted/founder viruses was greater in IDUs (range, 1 to 16 viruses; median, 3) than in either heterosexuals (range, 1 to 6 viruses; median, 1) or MSM (range, 1 to 10 viruses; median, 1). The finding of larger numbers of transmitted/founder viruses in IDUs was not simply the result of more intensive sampling, since the numbers of sequences analyzed in all studies were comparable. Moreover, it is notable that in studies reported elsewhere, we sampled as many as 239 sequences by SGA or as many as 500,000 sequences by 454 pyrosequencing from four acutely infected MSM subjects and in each case found evidence of productive clinical infection by only a single virus (; W. Fischer, B. Keele, G. Shaw, and B. Korber, unpublished). These results thus suggest that IDUs may be infected by more viruses and by a greater range of viruses than is the case following mucosal transmission. On this count, our findings differ from those reported by Masharsky and coworkers for an IDU cohort from St. Petersburg, Russia (). Their study found a low frequency of multiple virus transmissions (31%), not significantly different from that of acutely infected heterosexuals, and a low number of transmitted/founder viruses (range, 1 to 3 viruses; median, 1). Because the SGA methods employed in both studies were identical, the numbers of sequences analyzed per subject were comparable (median of 25 sequences in Montreal versus 33 in St. Petersburg), and because the discriminating power of the SGA-direct sequencing method was sufficient to distinguish transmitted/founder viruses differing by as few as 3 nucleotides, or <0.1% of nucleotides (Fig. (Fig.2,2, compare lineages V4 and V5), it is unlikely that differences in the genetic diversity of HIV-1 in the two IDU populations explain the differences in findings between the two studies. Instead, we suspect that the explanation lies in the small cohort sizes (10 versus 13 subjects) and the particular risk behaviors of the IDUs in each cohort. The Russian cohort is heavily weighted toward heroine use, whereas the Montreal cohort is weighted toward injection cocaine use, the latter being associated with more frequent drug administration and the attendant infection risks of needle sharing ().

TABLE 2.

Multiplicity of HIV-1 infection in IDU, heterosexual, and MSM subjects

Cohort	Reference	Virus subtype	Total no. of subjects	Single-variant transmission		Multiple-variant transmission		P value	Odds ratio	95% CI	Median	Range
Cohort	Reference	Virus subtype	Total no. of subjects	No. of subjects	% of total	No. of subjects	% of total	P value	Odds ratio	95% CI	Median	Range
Heterosexuals	Keele et al. (18)	B	79	65	82.30	14	17.70				1	1-4
	Abrahams et al. (1)	C	69	54	78.30	15	21.70				1	1-5
	Haaland et al. (13)	A or C	27	22	81.50	5	18.50				1	1-6
	Total		175	141	80.60	34	19.40	0.008^a	6.14	1.37-31.27	1	1-6
MSM	Keele et al. (18)	B	22	13	59.10	9	40.90				1	1-6
	Li et al. (24)	B	28	18	64.30	10	35.70				1	1-10
	Total		50	31	62.00	19	38.00	0.294^b	2.41	0.50-13.20	1	1-10
IDUs	Bar	B	10	4	40.00	6	60.00				3	1-16

Open in a separate window^aFisher''s exact test of multiple-variant transmission in heterosexuals versus in IDUs.^bFisher''s exact test of multiple-variant transmission in MSM versus in IDUs.The results from the present study indicate that transmission of HIV-1 to IDUs can be associated with a high frequency of multiple-variant transmission and a broad range in the numbers of transmitted viruses. This wide variation in the multiplicity of HIV-1 infection in IDUs is likely due to the absence of a mucosal barrier to virus transmission (, ) and differences in the virus inocula (, , , ). The findings substantiate concerns raised in recent HIV-1 vaccine efficacy trials that different vaccine candidates may be more efficacious in preventing infection by some exposure routes than by others (, , , ). They further suggest that biological comparisons of molecularly cloned transmitted/founder viruses responsible for vaginal, rectal, penile, and intravenous infection could facilitate a mechanistic understanding of HIV-1 transmission and vaccine prevention (, ). 相似文献