William R. Engels

Contributing software to the internet: the amplify program

Abstract The Internet provides a good way to distribute software to a wide range of potential users. Your program becomes instantly available to anyone who cares to download it. This note is about Amplify , a program I wrote a little more than a year ago, and about the more general topic of distributing scientific software.

The Drosophila Mre11/Rad50 Complex Is Required to Prevent Both Telomeric Fusion and Chromosome Breakage

The MRN complex consists of the two evolutionarily conserved components Mre11 and Rad50 and the third less-conserved component Nbs1/Xrs2. This complex mediates telomere maintenance in addition to a variety of functions in response to DNA double-strand breaks, including homologous recombination, nonhomologous end joining (NHEJ), and activation of DNA damage checkpoints. Mutations in the Mre11 gene cause the human ataxia-telangiectasia-like disorder (ATDL). Here, we show that null mutations in the Drosophila mre11 and rad50 genes cause both telomeric fusion and chromosome breakage. Moreover, we demonstrate that these mutations are in the same epistasis group required for telomere capping and mitotic chromosome integrity. Using an antibody against Rad50, we show that this protein is uniformly distributed along mitotic chromosomes, and that Rad50 is unstable in the absence of its binding partner Mre11. To define the roles of rad50 and mre11 in telomere protection, mutant chromosome preparations were immunostained for both HP1 and HOAP, two proteins that protect Drosophila telomeres from fusion. Cytological analysis revealed that mutations in rad50 and mre11 drastically reduce accumulation of HOAP and HP1 at telomeres. This suggests that the MRN complex protects Drosophila telomeres by facilitating recruitment of HOAP and HP1 at chromosome ends.

Differential usage of alternative pathways of double-strand break repair in Drosophila.

Double-strand DNA breaks can be repaired by any of several alternative mechanisms that differ greatly in the nature of the final repaired products. We used a reporter construct, designated “Repair reporter 3” (Rr3), to measure the relative usage of these pathways in Drosophila germ cells. The method works by creating a double-strand break at a specific location such that expression of the red fluorescent protein, DsRed, in the next generation can be used to infer the frequency at which each pathway was used. A key feature of this approach is that most data come from phenotypic scoring, thus allowing large sample sizes and considerable precision in measurements. Specifically, we measured the proportion of breaks repaired by (1) conversion repair, (2) nonhomologous end joining (NHEJ), or (3) single-strand annealing (SSA). For conversion repair, the frequency of mitotic crossing over in the germ line indicates the relative prevalence of repair by double Holliday junction (DHJ) formation vs. the synthesis-dependent strand annealing (SDSA) pathway. We used this method to show that breaks occurring early in germ-line development were much more frequently repaired via single-strand annealing and much less likely to be repaired by end joining compared with identical breaks occurring later in development. Conversion repair was relatively rare when breaks were made either very early or very late in development, but was much more frequent in between. Significantly, the changes in relative usage occurred in a compensatory fashion, such that an increase in one pathway was accompanied by decreases in others. This negative correlation is interpreted to mean that the pathways for double-strand break repair compete with each other to handle a given breakage event.

Identifying P factors in Drosophila by means of chromosome breakage hotspots

A syndrome of germline abnormalities in Drosophila melanogaster called hybrid dysgenesis is thought to be caused by transposable genetic elements known as P factors. Several lines of evidence presented here show that the chromosomal positions of at least some P factors can be identified as points of frequent chromosome breakage (hotspots). Starting with a strain (pi 2) in which four hotspots had been identified on the X chromosome, we found individual hotspots vanished when their part of the chromosome was replaced by the homologous part from a strain known to lack P factors. All hotspots in the non-substituted parts of the chromosome remained functional, indicating that they can act autonomously. We also observed a new breakage site coinciding with the appearance of an unstable mutation at the singed bristle locus (snW). This mutation was dysgenesis-induced, and previous genetic evidence suggested that it was caused by the insertion of a P factor at that locus. We also present preliminary evidence for rapid scrambling of the positions of hotspots under certain conditions, and we describe a new procedure for efficiently determining the positions of hotspots on a given chromosome.

Base substitutions, length differences and DNA strand asymmetries in the human Gγ and Aγ fetal globin gene region

Abstract We have studied differences arising subsequent to the 5 kilobase pair (kb) duplication that led to the human G γ and A γ fetal globin genes. The local occurrence of base substitutions in the duplicated 5 kb region correlates positively with the local AT base pair content. This correlation also occurs in two mouse β-globin genes and in two mouse immunoglobulin genes. The relationship is valid for transcribed or nontranscribed DNA and for DNA that contains only coding sequences. Length differences in the fetal globin duplicated regions correlate positively with the occurrence of short direct repeats of ≥5 base pairs. Path analysis of the interrelationships of base composition, base substitutions, repeats and length differences provides an integrated view of the relative effects on chromosomal changes of these variables and of selection. The distributions along the chromosome of simple sequences and of base compositions show highly significant local asymmetries between the transcribed and nontranscribed strands of the DNA, which permit us to divide the fetal globin gene region into chromosomal domains. Comparable domains are present in DNA from other sources, including the mammalian viruses SV40 and polyoma virus strain A-2 in which some of the domains appear related to discrete functions.

Spread of P transposable elements in inbred lines of Drosophila melanogaster.

Publisher Summary This chapter describes experiments in which single P elements were observed to spread in M strain genomes. Some of the uncertainties inherent in the previous experimental designs were avoided through the use of in situ hybridization of P-element sequences to polytene chromosomes, as opposed to genomic Southern blots of massextracted DNA. In addition, to follow the chromosomes as closely as possible, the populations were maintained by brother-sister matings, as opposed to mass transfers, during the generations when P-element sites were being monitored. It is considered that control over the population size and structure is a critical feature in this experiment, since the data indicate that these parameters are important factors in determining the fate of invading P elements. The approach discussed in the chapter does not allow detection of heterochromatic P elements. However, it affords much higher resolution, and is less likely to obscure any events that require observation of individual genomes.

Exact Tests for Hardy–Weinberg Proportions

Exact conditional tests are often required to evaluate statistically whether a sample of diploids comes from a population with Hardy–Weinberg proportions or to confirm the accuracy of genotype assignments. This requirement is especially common when the sample includes multiple alleles and sparse data, thus rendering asymptotic methods, such as the common χ2-test, unreliable. Such an exact test can be performed using the likelihood ratio as its test statistic rather than the more commonly used probability test. Conceptual advantages in using the likelihood ratio are discussed. A substantially improved algorithm is described to permit the performance of a full-enumeration exact test on sample sizes that are too large for previous methods. An improved Monte Carlo algorithm is also proposed for samples that preclude full enumeration. These algorithms are about two orders of magnitude faster than those currently in use. Finally, methods are derived to compute the number of possible samples with a given set of allele counts, a useful quantity for evaluating the feasibility of the full enumeration procedure. Software implementing these methods, ExactoHW, is provided.

Template disruptions and failure of double Holliday junction dissolution during double-strand break repair in Drosophila BLM mutants

Previous biochemical studies of the BLM gene product have shown its ability in conjunction with topoisomerase IIIα to resolve double Holliday structures through a process called “dissolution.” This process could prevent crossing over during repair of double-strand breaks. We report an analysis of the Drosophila BLM gene, DmBlm, in the repair of double-strand breaks in the premeiotic germ line of Drosophila males. With a repair reporter construct, Rr3, and other genetic tools, we show that DmBlm mutants are defective for homologous repair but show a compensating increase in single-strand annealing. Increases of 40- to 50-fold in crossing over and flanking deletions also were seen. Perhaps most significantly, the template used for homologous repair in DmBlm mutants is itself subject to deletions and complex rearrangements. These template disruptions are indicative of failure to resolve double Holliday junctions. These findings, along with the demonstration that a weak allele of topoisomerase IIIα has some of the same defects as DmBlm, support the dissolution model. Finally, an analysis of DmBlm mutants in conjunction with mus81 or spnA (Rad51) reveals a second function of BLM distinct from the repair of induced double-strand breaks and possibly related to maintenance of replication forks.

High levels of recombination induced by homologous P elements in Drosophila melanogaster

SummaryP element transposons in Drosophila melanogaster are capable of mobilizing incomplete P elements elsewhere in the genome, and of inducing recombination. This recombination is usually only of the order of 1% or less. We show that two P elements, located at exactly homologous sites, induce levels of recombination of 20% or higher. The recombination appears to be exact, as determined by the lack of phenotypic effects in recombinant products and the lack of size changes detectable by Southern hybridization. Female recombination is increased, but to a lesser extent than male recombination. Somatic recombination levels are also elevated. Alternative explanations for the high recombination levels are given in terms of the consequences of repair of an excision site and in terms of recombination as part of the replicative transposition process.

A Third Link connecting Aging with Double Strand Break Repair

Until recently, the connection between aging and DNA repair has rested on two classes of observation. First, DNA damage and unrepaired double-strand breaks (DSBs) accumulate with age. Second, several defects in DNA repair genes are associated with early onset of age-related diseases and other signs of premature aging. Now, a third link has emerged: The mechanisms by which cells repair DSB damage can change dramatically with age, shifting from simpler end-joining processes in younger organisms to homologous mechanisms in which missing genetic information is restored through use of a template. So far this third link between aging and DNA repair has only been observed in a small number of experimental systems, and cannot yet claim the generality of the other two. Here we review the evidence for this phenomenon and present new data testing models for the underlying causes. If the generality of age-related changes in DSB repair pathway usage can be established, it will provide a new insight into the underlying molecular basis of aging and how evolution has shaped these processes.