Regulation of IS1 transposition by the insA gene product

The IS1 element contains two adjacent genes called insA and insB, both required for IS1 transposition and IS1-mediated plasmid cointegration. These two genes are transcribed polycistronically from the promoter in the left terminal inverted repeat of IS1 (insL). We constructed overexpression systems of these genes with the tac promoter, which are regulated by an exogenous inducer, isopropyl-beta-D-thiogalactopyranoside (IPTG). Then we have examined, under various conditions of induction with IPTG, how overexpression of these genes affects IS1 transposition, using an assay based on plasmid cointegration. When the insA and insB genes were organized identically to the wild-type IS1 genes and simultaneously expressed using low concentrations of IPTG, activity of a mutant IS1 in cis was restored, but not in trans. Higher IPTG concentrations resulted in lower transposition activity. Expression in trans of insA and insB results in a 50 to 100-fold reduction of the frequency of cointegration mediated by wild-type IS1. Such a reduction is also observed when only the insA gene is overexpressed in trans. Overexpression of either mutant insA or insB does not affect the cointegration event. Tests with the insA-lacZ fusion gene showed that the InsA product inhibits the expression of IS1 genes directed by its own promoter in insL. These results suggest that the InsA product regulates IS1 transposition by inhibiting expression of IS1 transposition genes in addition to acting as part of a transposase complex.     

Nonallelic gene conversion is not GC-biased in Drosophila or primates

Gene conversion is the unidirectional transfer of genetic information between allelic (orthologous) or nonallelic (paralogous) DNA segments. Recently, there has been much interest in understanding how gene conversion shapes the nucleotide composition of the genomic landscape. A widely held hypothesis is that gene conversion is universally GC-biased. However, direct experimental evidence of this hypothesis is limited to a single study of meiotic crossovers in yeast. Although there have been a number of indirect studies of gene conversion, evidence of GC-biased replacements gathered from such studies can also be attributed to positive selection, which has the same evolutionary dynamics as biased gene conversion. Here, we apply a direct phylogenetic approach to examine nucleotide replacements produced by nonallelic gene conversion in Drosophila and primate genomes. We find no evidence for GC-biased gene conversion in either lineage, suggesting that previously observed GC biases may be due to positive selection rather than to biased gene conversion.     

Unexpectedly rapid IS1 transposition into an Arabidopsis chromatin remodeling gene

Common cloning is often associated with instability of certain classes of DNA. Here we report on IS1 transposition as possible source of such instability. During the cloning of Arabidopsis thaliana gene into commercially available vector maintained in widely used Escherichia coli host the insertion of complete IS1 element into the intron of cloned gene was found. The transposition of the IS1 element was remarkably rapid and is likely to be sequence-specific. The use of E. coli strains that lower the copy number of vector or avoiding the presence of the problematic sequence is a solution to the inadvertent transposition of IS1. The transposition of IS1 is rare but it can occur and might confound functional studies of a plant gene.     

L1 retrotransposon-mediated stable gene silencing

RNA interference (RNAi) is widely used for functional studies and has been proposed as a potential therapeutic agent. Current RNAi systems are largely efficient, but have limitations including transient effect, the need for viral handling and potential insertional mutations. Here, we describe a simple L1 retrotransposon-based system for the delivery of small interfering RNA (siRNA) and stable silencing in human cells. This system demonstrated long-term siRNA expression and significant reduction in both exogenous and endogenous gene expression by up to 90%. Further characterization indicated that retrotransposition occurred in a controlled manner such that essentially only one RNAi-cassette was integrated into the host genome and was sufficient for strong interference. Our system provides a novel strategy for stable gene silencing that is easy and efficient, and it may have potential applications for ex vivo and in vivo molecular therapy.     

Insulin sensitivity and big ET-1 conversion to ET-1 after ETA- or ETB-receptor blockade in humans.

Cardiovascular diseases are characterized by insulin resistance and elevated endothelin (ET)-1 levels. Furthermore, ET-1 induces insulin resistance. To elucidate this mechanism, six healthy subjects were studied during a hyperinsulinemic euglycemic clamp during infusion of (the ET-1 precursor) big ET-1 alone or after ET(A)- or ET(B)-receptor blockade. Insulin levels rose after big ET-1 with or without the ET(B) antagonist BQ-788 (P < 0.05) but were unchanged after the ET(A) antagonist BQ-123 + big ET-1. Infused glucose divided by insulin fell after big ET-1 with or without BQ-788 (P < 0.05). Insulin and infused glucose divided by insulin values were normalized by ET(A) blockade. Mean arterial blood pressure rose during big ET-1 with or without BQ-788 (P < 0.001) but was unchanged after BQ-123. Skeletal muscle, splanchnic, and renal blood flow responses to big ET-1 were abolished by BQ-123. ET-1 levels rose after big ET-1 (P < 0.01) in a similar way after BQ-123 or BQ-788, despite higher elimination capacity after ET(A) blockade. In conclusion, ET-1-induced reduction in insulin sensitivity and clearance as well as splanchnic and renal vasoconstriction are ET(A) mediated. ET(A)-receptor stimulation seems to inhibit the conversion of big ET-1 to ET-1.     

Orientation of IS50 transposase gene and IS50 transposition.

Reversal of transposase gene orientation with respect to the nonidentical ends of IS50 strongly decreased IS50 transposition in both Dam- and Dam+ hosts. In either orientation, IS50 transposase expression was unaffected. These effects were independent of the surrounding DNA context. This shows that the efficiency of IS50 transposition is dependent on transposase gene orientation. The transposition frequencies of transposons utilizing inverted IS50 inside ends (IE), IE-IE transposons, were lower than either outside end (OE)-IE or OE-OE transposons.     

Conditional conversion of ES cells to skeletal muscle by an exogenous MyoD1 gene.

A variety of differentiated cell types can be converted to skeletal muscle cells following transfection with the myogenic regulatory gene MyoD1. To determine whether multipotent embryonic stem (ES) cells respond similarly, cultures of two ES cell lines were electroporated with a MyoD1 cDNA driven by the beta-actin promoter. All transfected clones, carrying a single copy of the exogenous gene, expressed high levels of MyoD1 mRNA. Surprisingly, although maintained in mitogen-rich medium, this ectopic expression was associated with a transactivation of the endogenous myogenin and myosin light chain 2 gene but not the endogenous MyoD1, MRF4, Myf5, the skeletal muscle actin, or the myosin heavy chain genes. Preferential myogenesis and the appearance of contracting skeletal muscle fibers were observed only when the transfected cells were allowed to differentiate in vitro, via embryoid bodies, in low-mitogen-containing medium. Myogenesis was associated with the activation of MRF4 and Myf5 genes and resulted in a significant increase in the level of myogenin mRNA. Not all cells were converted to skeletal muscle cells, indicating that only a subset of stem cells can respond to MyoD1. Moreover, the continued expression of the introduced gene was not required for myogenesis. These results show that ES cells can respond to MyoD1, but environmental factors control the expression of its myogenic differentiation function, that MyoD1 functions in ES cells even under environmental conditions that favor differentiation is not dominant (incomplete penetrance), that MyoD1 expression is required for the establishment of the myogenic program but not for its maintenance, and that the exogenous MyoD1 gene can trans-activate the endogenous myogenin and MLC2 genes in undifferentiated ES cells.     

Alternative use of a mini exon of the L1 gene affects L1 binding to neural ligands.

Neural cell adhesion molecule L1 is a cell surface glycoprotein required for the correct development of the nervous system. L1 exists as two isoforms encoded by mRNA species that either collectively incorporate or exclude exons 2 and 27. Neurons utilize only the full-length isoform, whereas Schwann cells, kidney cells, and blood lymphocytes only express the short form of L1. Still other cells, oligodendrocytes, regulate L1 isoform expression in a maturation-dependent manner. The RSLE motif encoded by exon 27 is known to have a role in clathrin-mediated endocytosis of L1, but the function of the exon 2-encoded motif (YEGHHV) is unknown. Here we show that this motif is required for the optimal binding of L1 to several neural ligands and is likely to be important for nervous system development. Thus, alternative use of exon 2 is a mechanism for regulating ligand interactions with L1.     

Transposon Tn1 intra-molecular transposition

Summary A system for the direct selection of intra- and inter-molecular transposition events has been used to show that intra-molecular transposition of Tn1 generates deletions and inversions and requires the tnpA but not the tnpR gene product, as predicted by current models of transposition. Intra-molecular Tn1 transposition is much less limited by transposition immunity than inter-molecular transposition, and occurs at frequencies comparable to those for inter-molecular transposition. The selection system, which uses the bacteriophage cI-P_R region as a target can be used to select, quantify, and characterize any spontaneous or induced mutations.     

Detecting gene conversion: primate visual pigment genes.

The effects of gene conversion can be detected in the DNA sequences of multigene families. We develop a permutation test of the significance of patterns of sequence mismatches, and apply it to the sequences of the red- and green-sensitive visual pigment genes of human and the diana monkey. Whereas conventional tests of the rate of sequence divergence are equivocal, the permutation test convincingly excludes divergence in the absence of gene conversion (p = 10(-6)).     

Extrachromosomal and chromosomal gene conversion in mammalian cells.

We constructed substrates to study gene conversion in mammalian cells specifically without the complication of reciprocal recombination events. These substrates contain both an insertion mutation of the neomycin resistance gene (neoX) and an internal, homologous fragment of the neo gene (neo-526), such that gene conversion from neo-526 to neoX restores a functional neo gene. Although two reciprocal recombination events can also produce an intact neo gene, these double recombination events occur much less frequently that gene conversion in mammalian cells, We used our substrates to characterize extrachromosomal gene conversion in recombination-deficient bacteria and in monkey COS cells. Chromosomal recombination was also studied after stable integration of these substrates into the genome of mouse 3T6 cells. All extrachromosomal and chromosomal recombination events analyzed in mammalian cells resulted from gene conversion. Chromosomal gene conversion events occurred at frequencies of about 10(-6) per cell generation and restored a functional neo gene without overall effects on sequence organization.     

Accuracy of intrachromosomal gene conversion in mouse cells.

Results of several recent studies suggest that homologous recombination and related processes in mammalian cells are highly mutagenic. We have examined the products of intrachromosomal gene conversion events that encompassed the last intron of the chicken thymidine kinase gene. Following plasmid rescue and DNA sequencing, we find no mutations associated with twenty conversion events representing 5380 total base pairs of which 2414 base pairs are intron sequence. Based on these studies we conclude that intrachromosomal gene conversion in mouse cells is not a highly mutagenic process but rather it operates with fidelity.     

High-frequency germ line gene conversion in transgenic mice.

Gene conversion is the nonreciprocal transfer of genetic information between two related genes or DNA sequences. It can influence the evolution of gene families, having the capacity to generate both diversity and homogeneity. The potential evolutionary significance of this process is directly related to its frequency in the germ line. While measurement of meiotic inter- and intrachromosomal gene conversion frequency is routine in fungal systems, it has hitherto been impractical in mammals. We have designed a system for identifying and quantitating germ line gene conversion in mice by analyzing transgenic male gametes for a contrived recombination event. Spermatids which undergo the designed intrachromosomal gene conversion produce functional beta-galactosidase (encoded by the lacZ gene), which is visualized by histochemical staining. We observed a high incidence of lacZ-positive spermatids (approximately 2%), which were produced by a combination of meiotic and mitotic conversion events. These results demonstrate that gene conversion in mice is an active recombinational process leading to nonparental gametic haplotypes. This high frequency of intrachromosomal gene conversion seems incompatible with the evolutionary divergence of newly duplicated genes. Hence, a process may exist to uncouple gene pairs from frequent conversion-mediated homogenization.     

Statistical methods of DNA sequence analysis: detection of intragenic recombination or gene conversion

Simple but exact statistical tests for detecting a cluster of associated nucleotide changes in DNA are presented. The tests are based on the linear distribution of a set of s sites among a total of n sites, where the s sites may be the variable sites, sites of insertion/deletion, or categorized in some other way. These tests are especially useful for detecting gene conversion and intragenic recombination in a sample of DNA sequences. In this case, the sites of interest are those that correspond to particular ways of splitting the sequences into two groups (e.g., sequences A and D vs. sequences B, C, and E-J). Each such split is termed a phylogenetic partition. Application of these methods to a well-documented case of gene conversion in human gamma-globin genes shows that sites corresponding to two of the three observed partitions are significantly clustered, whereas application to hominoid mitochondrial DNA sequences--among which no recombination is expected to occur--shows no evidence of such clustering. This indicates that clustering of partition-specific sites is largely due to intragenic recombination or gene conversion. Alternative hypotheses explaining the observed clustering of sites, such as biased selection or mutation, are discussed.      

dnaA, an essential host gene, and Tn5 transposition.

Mutations in dnaA, an essential gene in Escherichia coli, decrease the frequency of transposition of Tn5. An insertion mutation in the dnaA gene does not affect Tn5 gene expression. Therefore, the DnaA protein plays a role either in the transposition reaction itself or in some type of cellular regulation of transposition. Analysis of a mutation in the DnaA box, found at the outside end of IS50, is consistent with a direct interaction of the protein through these bases. IS50 transposition, which utilizes only one end containing a DnaA box, is not affected by dnaA mutations. Overproduction of the DnaA protein does not increase transposition frequencies in wild-type cells, even when the transposase is also overproduced.     

Surprising fitness consequences of GC-biased gene conversion. II. Heterosis

Heterosis is a widespread phenomenon corresponding to the increase in fitness following crosses between individuals from different populations or lines relative to their parents. Its genetic basis has been a topic of controversy since the early 20th century. The masking of recessive deleterious mutations in hybrids likely explains a substantial part of heterosis. The dynamics and consequences of these mutations have thus been studied in depth. Recently, it was suggested that GC-biased gene conversion (gBGC) might strongly affect the fate of deleterious mutations and may have significant fitness consequences. gBGC is a recombination-associated process mimicking selection in favor of G and C alleles, which can interfere with selection, for instance by increasing the frequency of GC deleterious mutations. I investigated how gBGC could affect the amount and genetic structure of heterosis through an analysis of the interaction between gBGC and selection in subdivided populations. To do so, I analyzed the infinite island model both by numerical computations and by analytical approximations. I showed that gBGC might have little impact on the total amount of heterosis but could greatly affect its genetic basis.