Endogenous retrovirus expression in stimulated murine lymphocytes. Identification of a new locus controlling mitogen induction of a defective virus.

Germ line DNA from all strains of mice contains numerous endogenous retroviruses. One of these viruses, a virus with xenotropic host range is induced from lymphocytes of most strains by treatment with B cell mitogens. Virus induction is amplified by 5-bromo-2'-deoxyuridine (BrdU) treatment. We report here studies of the genetic control of retrovirus induction from lymphocytes in crosses between BALB/cTif mice and noninducible 129/Rrj mice. We identify a novel locus, Bdv-1, which controls the expression of a reverse transcriptase-positive, defective retrovirus in BALB/cTif lymphocytes. In addition, we confirm previous reports that xenotropic virus is controlled by a locus, Bxv-1, mapping to chromosome 1. The two loci are nonlinked and respond differently to inducing stimuli. Bxv-1 is induced mainly by BrdU and only marginally by mitogen; in contrast, Bdv-1 is induced by mitogen and BrdU has little effect. The induction of these two loci is discussed with respect to B cell differentiation.

Endogenous retroviruses and the human germline.

The human genome is rife with the proviral remains of many ancient retroviruses. The past year has seen significant progress in understanding the structure, distribution and potential function of many of these elements. Although hypotheses concerning the potential effects of these elements are common, however, incisive experiments to test any functions remain much less so.

Endogenous retroviruses: still active after all these years?

Embedded in the genomes of all vertebrates are the proviral remnants of previous retroviral infections. Although the overwhelming majority has suffered inactivating mutations, current research suggests that members of one family of human retroelements may still be capable of movement.

REPuter: the manifold applications of repeat analysis on a genomic scale.

The repetitive structure of genomic DNA holds many secrets to be discovered. A systematic study of repetitive DNA on a genomic or inter-genomic scale requires extensive algorithmic support. The REPuter program described herein was designed to serve as a fundamental tool in such studies. Efficient and complete detection of various types of repeats is provided together with an evaluation of significance and interactive visualization. This article circumscribes the wide scope of repeat analysis using applications in five different areas of sequence analysis: checking fragment assemblies, searching for low copy repeats, finding unique sequences, comparing gene structures and mapping of cDNA/EST sequences.

Endogenous retroviruses and the evolution of resistance to retroviral infection.

The current AIDS epidemic has rekindled interest in the evolution of retroviruses and the development of resistance to infection. Retroviruses and their vertebrate hosts have coexisted for millions of years, during which time a variety of host defence mechanisms has evolved. One repeated strategy is to use endogenous retroviruses to combat infection by their exogenous relatives.

A linkage map of endogenous murine leukemia proviruses.

Thirty endogenous proviruses belonging to the modified polytropic (Mpmv) class of murine leukemia virus (MLV) were identified by proviral-cellular DNA junction fragment segregation in several sets of recombinant inbred mice. Twenty-six Mpmv loci were mapped to chromosomal regions by matching proviral strain distribution patterns to those of previously assigned genes. Like other endogenous nonecotropic MLVs, Mpmv loci were present on several chromosomes in all strains examined. We pooled recombinant inbred strain linkage data from 110 MLV loci and selected marker genes in order to construct a chromosomal linkage map. Every mouse chromosome was found to harbor at least one proviral insertion, and several regions contained multiple integrations. However, the overall distribution of the 110 mapped proviruses did not deviate significantly from a random distribution. Because of their polymorphism in inbred strains of mice, and the ability to score as many as 57 proviruses per strain using only three hybridization probes, the nonecotropic MLVs mapped in common strains of mice offer a significant advantage over older methods (e.g., biochemical or individual restriction fragment polymorphisms) as genetic markers. These endogenous insertion elements should also be useful for assessing strain purity, and for studying the relatedness of common and not-so-common inbred strains.

Multiple sequence alignment with the Divide-and-Conquer method.

An improved algorithm for the simultaneous alignment of multiple protein and nucleic acid sequences, the Divide-and-Conquer Alignment procedure (DCA), is presented. The basic method described in Tönges,et al. (1996) (Tönges, U., Perrey, S.W., Stoye, J., Dress, A.W.M., 1996. A general method for fast multiple sequence alignment. Gene, 172, GC33-GC41) is generalized to align any number of sequences to work arbitrary (e.g. affine linear) gap penalty functions. Also, the practical efficiency of the method is improved so that families of more than 10 sequences can now be aligned simultaneously within a few seconds or minutes. After a brief description of the general method, we assess the time and memory requirements of our implementation of DCA. We present several examples showing that the program is able to deal with real-world alignment problems.

A general method for fast multiple sequence alignment.

We have developed a fast heuristic algorithm for multiple sequence alignment which provides near-to-optimal results for sufficiently homologous sequences. The algorithm makes use of the standard dynamic programming procedure by applying it to all pairs of sequences. The resulting score matrices for pair-wise alignment give rise to secondary matrices containing the additional charges imposed by forcing the alignment path to run through a particular vertex. Such a constraint corresponds to slicing the sequences at the positions defining that vertex, and aligning the remaining pairs of prefix and suffix sequences separately. From these secondary matrices, one can compute-for any given family of sequences-suitable positions for cutting all of these sequences simultaneously, thus reducing the problem of aligning a family of n sequences of average length l in a Divide and Conquer fashion to aligning two families of n sequences of approximately half that length. In this paper, we explain the method for the case of 3 sequences in detail, and we demonstrate its potential and its limits by discussing its behaviour for several test families. A generalization for aligning more than 3 sequences is lined out, and some actual alignments constructed by our algorithm for various user-defined parameters are presented.

Contig selection in physical mapping.

In physical mapping, one orders a set of genetic landmarks or a library of cloned fragments of DNA according to their position in the genome. Our approach to physical mapping divides the problem into smaller and easier subproblems by partitioning the probe set into independent parts (probe contigs). For this purpose we introduce a new distance function between probes, the averaged rank distance (ARD) derived from bootstrap resampling of the raw data. The ARD measures the pairwise distances of probes within a contig and smoothes the distances of probes across different contigs. It shows distinct jumps at contig borders. This makes it appropriate for contig selection by clustering. We have designed a physical mapping algorithm that makes use of these observations and seems to be particularly well suited to the delineation of reliable contigs. We evaluated our method on data sets from two physical mapping projects. On data from the recently sequenced bacterium Xylella fastidiosa, the probe contig set produced by the new method was evaluated using the probe order derived from the sequence information. Our approach yielded a basically correct contig set. On this data we also compared our method to an approach which uses the number of supporting clones to determine contigs. Our map is much more accurate. In comparison to a physical map of Pasteurella haemolytica that was computed using simulated annealing, the newly computed map is considerably cleaner. The results of our method have already proven helpful for the design of experiments aimed at further improving the quality of a map.

Genetics of endogenous murine leukemia viruses.

Inbred strains of mice contain in the genome 40-60 endogenous proviruses related to murine leukemia virus. To assess the genetic and pathogenic consequences of these to the host, we have developed a strategy to distinguish among the three different host-range subgroups--xenotropic, polytropic and modified polytropic--by using oligonucleotide probes specific for a polymorphic region in env. Each of these proteins detects a relatively small number of bands in a Southern blot, thus permitting us to enumerate all individual proviruses of this group. Using this approach, we have determined the distribution of different proviruses among inbred and recombinant inbred (RI) strains congenic or coisogenic for specific mutants. Using the RI results, we have been able to place over 100 proviruses on the mouse genetic map. A number of these are closely linked to well-characterized mutations, and we have been able to establish that at least one mutation, hr (hairless), was caused by a proviral insertion. If the other close linkages also prove to reflect causality, we estimate that up to 5% of recessive mutations in the mouse might be caused by insertion of proviruses of this group. Using a similar probe strategy, we have followed the evolution of murine leukemia viruses during spontaneous leukemogenesis in AKR mice. We have found that the final leukemogenic (MCF) virus is a recombinant of three different endogenous parents; an ecotropic virus, a polytropic virus that directs the gp70 region of env, and a xenotropic virus (identified as the inducible element Bxv-1) that directs the LTR. In addition to the recombinations, all such viruses also have a reduplication of the enhancer region of the LTR, compared to the endogenous parent. MCF viruses are created by these three genetic changes, which occur in a reproducible fashion and appear in the thymus between 10 and 14 weeks of age.

Endogenous retroviruses: a potential problem for xenotransplantation?

To overcome the shortage of suitable human donors for transplantation attention has recently turned to the possibility of using genetically modified pigs as a source of cells and organs. It has been suggested that such procedures might facilitate the introduction of novel retroviruses, normally resident in the pig germ line, into the human population (Stoye and Coffin, Nature Medicine 1: 1100, 1995). The consequences of such a transfer remain unclear; however, the demonstration that certain porcine cell lines express infectious retroviruses which can infect human cells (Patience et al., Nature Medicine 3: 282-286, 1997) emphasizes that there are grounds for practical concern. We have now cloned the envelope genes of the expressed viruses and are using these clones in studies of the interaction of the porcine viruses with their cellular receptors. We have also initiated studies of the inheritance and expression of human-tropic endogenous proviruses present in different pig populations. These studies reveal that at least two classes of human-tropic endogenous porcine retrovirus are widely distributed in pigs (Le Tissier et al., Nature 389: 681-681, 1997). The implications of our results for assessing the potential risk of retroviral transfer during xenotransplantation are discussed.

Molecular analysis of viable spontaneous and radiation-induced albino (c)-locus mutations in the mouse.

Thirty-one homozygous-viable, radiation-induced or spontaneous mutations at the albino (c) locus in mouse chromosome 7 were analyzed by Southern blot analysis with a tyrosine cDNA clone and with probes derived from the closely linked proviral integration sites Pmv-31 and Emv-23, which flank the tyrosinase gene on the proximal and distal sides, respectively. Thirteen of 27 radiation-induced and one of four spontaneous mutations manifested deletions or rearrangements for the tyrosinase gene. The sizes of four deletions found to break within the tyrosinase gene itself were estimated to be < or = 36 kb, < or = 40 kb, approximately 260 kb, and approximately 480 kb. Two homozygous-viable deletions were found to include flanking proviral loci, suggesting that they could be from 1500-2000 kb in length, if not longer. The existence of these very large, homozygous-viable deletions suggests that the one-to-two megabases including and surrounding the c locus harbor no genes essential for normal viability or fertility, although genes controlling more subtle (or "nonessential") phenotypes are likely to be present. These data thus provide some insight into the molecular structure of a number of viable c-locus mutations, whose nature could not be predicted solely on the basis of genetic analysis, as could be done for either lethal or reduced-pigment c mutations.

Fv1, the mouse retrovirus resistance gene.

A number of genes which affect the susceptibility of mice to infection by retroviruses have been described. One of the most interesting of these genes is Fv1 (Friend virus susceptibility 1), which acts at a stage in the retroviral life-cycle following virus entry into the cell but prior to integration and formation of proviral structures. A detailed understanding of the mode of action of Fv1 might be expected to shed fresh light on early steps of the retroviral replication, although progress has been slow in this area due to uncertainty about the nature of the Fv1 gene. The recent cloning of Fv1 by a positional approach fills this gap in current knowledge. Fv1 appears to be derived from a fragment of a retroviral genome, an observation that may suggest novel approaches to the control of retroviral replication.

Intracisternal A-type particle elements as genetic markers: detection by repeat element viral element amplified locus-PCR.

We describe a novel, PCR-based technique termed REVEAL-PCR for examining the inheritance of intracisternal A-type particles (IAP). Amplifications use an unlabeled primer to SINE repeats and a radiolabeled primer to the IAP long terminal repeat; labeled products, which can be resolved on sequencing gels, are formed when IAPs lie in proximity to SINEs. With this technique we have identified a total of 124 polymorphisms in the BXH and CXS recombinant inbred strains. We suggest that this method will be equally applicable for examining other gene families present at around a thousand copies per genome.

The four classes of endogenous murine leukemia virus: structural relationships and potential for recombination.

The process by which leukemogenic viruses are generated during the lifetime of certain strains of mice is poorly understood. We have therefore set out to define all the murine leukemia virus-related endogenous proviruses of HRS/J mice. We have cloned 34 different proviral fragments and their flanking cellular sequences. These have been characterized by restriction enzyme analysis, by fingerprinting in vitro-synthesized RNA, and by DNA sequencing. We conclude that all the proviruses can be assigned into one of four different classes: the previously characterized ecotropic, xenotropic, and polytropic viruses, as well as a new class we have termed modified polytropic viruses. The xenotropic, polytropic, and modified polytropic classes are closely related to one another, but as a group they differ considerably from the ecotropic class. Sequence analyses show that both polytropic and modified polytropic sequences can contribute env sequences to recombinant viruses.

Polymorphism of murine endogenous proviruses revealed by using virus class-specific oligonucleotide probes.

Inbred mice contain three classes of endogenous nonecotropic murine leukemia virus-related sequences, namely xenotropic, polytropic, and modified polytropic proviruses. Oligonucleotide probes specific for the three different classes were prepared and used to examine the diversity of endogenous sequences present in eight different strains of mice: HRS/J, BALB/cJ, A/J, AKR/J, C57BL/6J, DBA/2J, C57L/J, and C3H/HeJ. A high degree of polymorphism was observed. Overall, the strains showed between 17% (A/J and HRS/J) and 65% (C57BL/6J and C57L/J) shared proviruses, and only four proviruses were present in all eight strains. The similarity among the strains is due in part to the few proviruses present in all of the strains but also represents the independent assortment of a limited set of proviruses. These oligonucleotides provide a basis for determining the stability, distribution, and mutagenic potential of nonecotropic proviruses within the mouse genome.

An iterative method for faster sum-of-pairs multiple sequence alignment.

MOTIVATION: Multiple sequence alignment is an important tool in computational biology. In order to solve the task of computing multiple alignments in affordable time, the most commonly used multiple alignment methods have to use heuristics. Nevertheless, the computation of optimal multiple alignments is important in its own right, and it provides a means of evaluating heuristic approaches or serves as a subprocedure of heuristic alignment methods. RESULTS: We present an algorithm that uses the divide-and-conquer alignment approach together with recent results on search space reduction to speed up the computation of multiple sequence alignments. The method is adaptive in that depending on the time one wants to spend on the alignment, a better, up to optimal alignment can be obtained. To speed up the computation in the optimal alignment step, we apply the alpha(*) algorithm which leads to a procedure provably more efficient than previous exact algorithms. We also describe our implementation of the algorithm and present results showing the effectiveness and limitations of the procedure.