Isolation of human germ-line DNA suitable for recombinant DNA studies

Dna from human sperm cells can be isolated with size and purity sufficient for use in recombinant DNA research. The DNA averages 100 000 base pairs (bp) in size (about 70.10(6) daltons) and is free of somatic cell, bacterial, and viral DNA. It can therefore be cloned under P2 + EK2 conditions as stipulated in the 1978 NIH Guidelines for recombinant DNA research.     

Interspersed DNA element restricted to a specific group of telomeres in the dipteran Chironomus pallidivittatus.

Telomeres in the dipteran Chironomus pallidivittatus terminate with 340bp tandem DNA repeats belonging to different subfamilies with characteristic intertelomeric distribution. We have now found, interspersed between such repeats, a composite element of approx. 1400bp present in two similar size variants, with several components of nontelomeric origin. There were about 50 copies of the element, predominantly or exclusively present in a previously defined group of telomeres, characterized by a unique set of telomeric tandem repeat subfamilies. Elements were integrated at irregular distances from each other, and intervening telomeric tandem repeat DNA was variable in composition. Nevertheless, the flanks immediately surrounding the elements were identical for different elements; in other words, there was a site-specific insertion. We suggest that this selective invasion of a small part of the genome by an interspersed, probably rapidly evolving element is best explained by repeated gene conversions.     

A selfish origin for recombination

Recent findings of molecular biology show that recombination is initiated by interactions between homologous chromosomes and that an allele can induce the initiation of recombination on the homolog. Since gene conversion at the site of initiation is strong enough to promote the transmission of that allele, recombination may be a way for a self-promoting element to spread, even if it gives no advantage to the individual or to the population. I develop a simple model and discuss available molecular evidence in support of this hypothesis. A consequent argument is that with asexual reproduction the evolution of recombination leads to an intragenomic conflict, and a possible outcome of this conflict may be the origin of sexual reproduction.     

Polyandry and the decrease of a selfish genetic element in a wild house mouse population

Despite deleterious effects on individuals, the t haplotype is a selfish genetic element present in many house mouse populations. By distorting the transmission ratio, +/t males transmit the t haplotype to up to 90% of their offspring. However, t/t individuals perish in utero. Theoretical models based on these properties predict a much higher t frequency than observed, leading to the t paradox. Here, we use empirical field data and theoretical approaches to investigate whether polyandry is a female counterstrategy against the negative fitness consequences of such distorters. We found a significant decrease of the t frequency over a period of 5.5 years that cannot be explained by the effect of transmission ratio distortion and recessive lethals, despite significantly higher life expectancy of +/t females compared to +/+ females. We quantified life-history data and homozygous and heterozygous fitness effects. Population subdivision and inbreeding were excluded as evolutionary forces influencing the t system. The possible influence of polyandry on the t system was then investigated by applying a stochastic model to this situation. Simulations show that polyandry can explain the observed t dynamics, making it a biologically plausible explanation for low t frequencies in natural populations in general.     

DNA and telomeres: beginnings and endings

How a cell deals with its DNA ends is a question that returns us to the very beginnings of modern telomere biology. It is also a question we are still asking today because it is absolutely essential that a cell correctly distinguishes between natural chromosomal DNA ends and broken DNA ends, then processes each appropriately - preserving the one, rejoining the other. Effective end-capping of mammalian telomeres has a seemingly paradoxical requirement for proteins more commonly associated with DNA double strand break (DSB) repair. Ku70, Ku80, DNA-PKcs (the catalytic subunit of DNA-dependent protein kinase), Xrcc4 and Artemis all participate in DSB repair through nonhomologous end-joining (NHEJ). Somewhat surprisingly, mutations in any of these genes cause spontaneous chromosomal end-to-end fusions that maintain large blocks of telomeric sequence at the points of fusion, suggesting loss or failure of a critical terminal structure, rather than telomere shortening, is at fault. Nascent telomeres produced via leading-strand DNA synthesis are especially susceptible to these end-to-end fusions, suggesting a crucial difference in the postreplicative processing of telomeres that is linked to their mode of replication. Here we will examine the dual roles played by DNA repair proteins. Our review of this rapidly advancing field primarily will focus on mammalian cells, and cannot include even all of this. Despite these limitations, we hope the review will serve as a useful gateway to the literature, and will help to frame the major issues in this exciting and rapidly progressing field. Our apologies to those whose work we are unable to include.     

Identification of a fully-functional hobo transposable element and its use for germ-line transformation of Drosophila.

The transposable element hobo can be mobilized to induce a variety of genetic abnormalities within the germ-line of Drosophila melanogaster. Strains containing hobos have 3.0 kb elements and numerous smaller derivatives of the element. By analogy with other transposable element systems, it is likely that only the 3.0 kb elements are capable of inducing hobo mobilization. Here, we report that a cloned 3.0 kb hobo, called HFL1, is able to mediate germ-line transformation and therefore is an autonomous (fully-functional) transposable element. Germ-line transformation was observed when HFL1 and a marked hobo element were co-injected into recipient embryos devoid of endogenous hobos. Integration did not occur in the absence of the 3.0 kb element. A single copy of the marked hobo transposon inserted at each site, and the target sites were widely distributed throughout the genome. Integration occurred at (or very near) the termini of hobo, without internal rearrangement of the hobo or marker gene sequences. The hobo transformation system will allow us to determine the structural and regulatory features of hobo responsible for its mobilization and will provide novel approaches for the molecular and genetic manipulation of the Drosophila genome.     

Homing endonuclease genes: the rise and fall and rise again of a selfish element

Homing endonuclease genes (HEGs) are selfish genetic elements that spread by first cleaving chromosomes that do not contain them and then getting copied across to the broken chromosome as a byproduct of the repair process. The success of this strategy will depend on the opportunities for homing--in other words, the frequency with which HEG(+) and HEG(-) chromosomes come into contact--which varies widely among host taxa. HEGs are also unusual in that the selection pressure for endonuclease function disappears if they become fixed in a population, which makes them susceptible to degeneration and imposes a need for regular horizontal transmission between species. HEGs will be selected to reduce the harm done to the host organism, and this is expected to influence the evolution of their sequence specificity and maturase functions. HEGs may also be domesticated by their hosts, and are currently being put to human uses.     

Introduction of a functional P element into the germ-line of Drosophila hawaiiensis

When a plasmid carrying a P-transposable element (derived from Drosophila melanogaster) is injected into young embryos of D. hawaiiensis, the P-element sequence from the plasmid transposes into the germ-line chromosomes. The introduction of this P element into D. hawaiiensis provides an opportunity to study the behavior of the transposable element in a novel context. Germ-line transposition and numerical increase of the P elements are readily detected in D. hawaiiensis. Thus these aspects of P-element function do not require chromosomal or cytoplasmic properties that are unique to D. melanogaster. Since D. hawaiiensis is among those Drosophila that are most distantly related to D. melanogaster, these results suggest that P-element-mediated transformation may function in many species.     

The yeast 2 micron plasmid: strategies for the survival of a selfish DNA

Summary The designation of the yeast 2 circle as a selfish DNA molecule has been confirmed by demonstrating that the plasmid is lost with exponential kinetics from haploid yeast populations grown in continuous culture. We show that plasmid-free yeast cells have a growth rate advantage of some 1.5%–3% over their plasmid-containing counterparts. This finding makes the ubiquity of this selfish DNA in yeast strains puzzling. Two other factors probably account for its survival. First, the rate of plasmid loss was reduced by allowing haploid populations to enter stationary phase periodically. Second, it was not possible to isolate a plasmid-free segregant from a diploid yeast strain. Competition experiments demonstrated that stability in a diploid is conferred at the level of segregation and that plasmid-free diploid cells are at a selective advantage compared with their plasmid-containing counterparts. Yeast cells in nature are usually homothallic and must frequently pass through both diploid and stationary phases. The 2 plasmid appears to have evolved a survival strategy which exploits these two features of its host's life cycle.     

Structure of the Drosophila HeT-A transposon: a retrotransposon-like element forming telomeres

Telomeres of Drosophila appear to be very different from those of other organisms. A transposable element, HeT-A, plays a major role in forming telomeres and may be the sole structural element, since telomerase-generated repeats are not found. HeT-A transposes only to chromosome ends. It appears to be a retrotransposon but has novel structural features, which may be related to its telomere functions. A consensus sequence from cloned HeT-A elements defines an element of 6 kb. The coding region has retrotransposon-like overlapping open reading frames (ORFs) with a –1 frameshift in a sequence resembling the frameshift region of the mammalian HIV-1 retrovirus. Both the HeT-A ORFs contain motifs suggesting RNA binding. HeT-A-specific features include a long non-coding region, 3 of the ORFs, which makes up about half of the element. This region has a regular array of imperfect sequence repeats and ends with oligo(A), marking the end of the element and suggesting a polyadenylated RNA transposition intermediate. This 3 repeat region may have a structural role in heterochromatin. The most distal part of each complete HeT-A on the chromosome, the region 5 of the ORFs, has unusual conserved features, which might produce a terminal structure for the chromosome.     

Short telomeres induce a DNA damage response in Saccharomyces cerevisiae

Telomerase-deficient Saccharomyces cerevisiae cells show a progressive decrease in telomere length. When grown for several days in log phase, the tlc1Delta cells initially display wild-type growth kinetics with subsequent loss of growth potential after which survivors are generated via RAD52-dependent homologous recombination. We found that chromosome loss in these telomerase-deficient cells only increased after a significant decline in growth potential of the culture. At earlier stages of growth, as the telomerase-deficient cells began to show loss of growth potential, the cells arrested in G2/M and showed RNR3 induction and Rad53p phosphorylation. These responses were dependent on RAD24 and MEC1, suggesting that short telomeres are recognized as DNA damage and signal G2/M arrest.     

Population biology of a selfish sex ratio distorting element in a booklouse (Psocodea: Liposcelis)

Arthropods harbour a variety of selfish genetic elements that manipulate reproduction to be preferentially transmitted to future generations. A major ongoing question is to understand how these elements persist in nature. In this study, we examine the population dynamics of an unusual selfish sex ratio distorter in a recently discovered species of booklouse, Liposcelis sp. (Psocodea: Liposcelididae) to gain a better understanding of some of the factors that may affect the persistence of this element. Females that carry the selfish genetic element only ever produce daughters, although they are obligately sexual. These females also only transmit the maternal half of their genome. We performed a replicated population cage experiment, varying the initial frequency of females that harbour the selfish element, and following female frequencies for 20 months. The selfish genetic element persisted in all cages, often reaching very high (and thus severely female‐biased) frequencies. Surprisingly, we also found that females that carry the selfish genetic element had much lower fitness than their nondistorter counterparts, with lower lifetime fecundity, slower development and a shorter egg‐laying period. We suggest that differential fitness plays a role in the maintenance of the selfish genetic element in this species. We believe that the genetic system in this species, paternal genome elimination, which allows maternal control of offspring sex ratio, may also be important in the persistence of the selfish genetic element, highlighting the need to consider species with diverse ecologies and genetic systems when investigating the effects of sex ratio manipulators on host populations.     

p53 engages the cGAS/STING cytosolic DNA sensing pathway for tumor suppression

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Give me a break: How telomeres suppress the DNA damage response

Linear organization of the genome requires mechanisms to protect and replicate chromosome ends. To this end eukaryotic cells evolved telomeres, specialized nucleoproteic complexes, and telomerase, the enzyme that maintains the telomeric DNA. Telomeres allow cells to distinguish chromosome ends from sites of DNA damage. In mammalian cells this is accomplished by a protein complex, termed shelterin, that binds to telomeric DNA and is able to shield chromosome ends from the DNA damage machinery. In recent years, we have seen major advances in our understanding of how this protein complex works due to the generation of mouse models carrying mutations of individual shelterin components. This review will focus on our current understanding of how the shelterin complex is able to suppress the DNA damage response pathways, and on the cellular and organismal outcomes of telomere dysfunction.