Genetic effects of injection of Rous sarcoma virus DNA into polar plasm of early Drosophila melanogaster embryos

Retroviral proviruses and the transposable elements of eukaryotic genomes are structurally similar. The biological significance of eukaryotic transposable elements has not been examined extensively but it is known that, like prokaryotic transposons, these elements can induce mutations in adjacent genes and cause their transposition. It is of interest to determine whether retroviral proviruses have the same mutagenic and gene transposing ability as transposable elements, particularly because the retrovirus genome is assumed to have originated from transposable elements of lower eukaryotes. The transfer of DNA sequences into animal zygotes or embryos by microinjection is a promising experimental approach for eluxidating their functions: when foreign DNAs were introduced into a mouse germ line, mutations were induced and at least in some mice, the mutation was caused by the insertion of a retroviral sequence. We have introduced Rous sarcoma virus (RSV) DNA into a germ line of Drosophila melanogaster, and describe here the resultant genetic effects.     

DNA sequence at the end of IS1 required for transposition

The insertion sequence IS1 belongs to a class of bacterial transposable genetic elements that can form compound transposons in which two copies of IS1 flank an otherwise non-transposable segment of DNA. IS1 differs from other known elements of this class (such as IS10, IS50 and IS903) in several respects. It is one of the smallest known insertion elements, exhibits a relatively complex array of open reading frames, is present in the chromosomes of various Enterobacteria, in some cases in many copies, and its insertion can result in the duplication of either 8 or 9 base pairs (bp) in the target DNA. Furthermore, although, like other members of the compound class, it seems to undergo direct transposition, IS1 also promotes replicon fusion (co-integrate formation) at a relatively high frequency. Like all other elements studied to date, the integrity of the extremities of IS1 are essential for efficient transposition. We have constructed a test system to determine the minimal DNA sequences at the extremities of IS1 required for transposition. Sequential deletions of the end sequences reveal that 21-25 bp of an isolated extremity are sufficient for transposition. A specific sequence 13-23 bp from the ends, defining the edge of the minimal sequence, is implicated as an essential site. The sites, symmetrically arrayed at both ends of IS1, correspond to the apparent consensus sequence of the known binding sites for the Escherichia coli DNA-binding protein (called integration host factor or IHF) which is required for the site-specific recombination that leads to integration of bacteriophage lambda into the bacterial genome. The sites at the ends of IS1 may thus bind a host protein, such as JHF or a related protein, that is involved in regulating the transposition apparatus.     

DNA sequences at the ends of transposon Tn5 required for transposition

Transposons are a class of genetic elements that can move from one site in a cell's genome to another independently of the cell's general recombination system. Little is known about the mechanism of transposition of compound transposons such as Tn5, but it is thought that a transposon-encoded protein (a transposase) must recognize the outer ends of the element and, together with host factors, catalyse the transfer of the internal DNA into a new site in a manner that may involve replication. It has previously been shown that the synthesis of an IS50R-encoded protein (protein 1) is an essential requirement for Tn5 transposition. Here we demonstrate that a structure containing only the outer 186 base pairs (bp) of both inverted repeats is capable of being efficiently complemented to transpose in Escherichia coli, provided IS50R is located close by on the same replicon. In addition, Bal31-generated deletions indicate that 16-18 bp of the outer end of IS50L are required for transposition. This 16-18-bp sequence contains the 8-9-bp small inverted repeat present at each end of IS50 plus a 9-bp sequence which is homologous to an interrelated sequence present in four copies in the chromosomal origin of replication in a variety of Gram-negative bacteria. This sequence organization suggests that the ends of Tn5 may function to provide a recognition site for the Tn5 transposase adjacent to a sequence recognized by the host replication system.     

A copia-like transposable element family in Arabidopsis thaliana

The fast generation time, small genome size and extensive genetic map of the crucifer Arabidopsis thaliana have made it the subject of an increasing number of studies in plant molecular genetics. As transposable elements have greatly facilitated genetic analysis in a variety of species, we have attempted to identify an endogenous A. thaliana transposable element. We report here the discovery of a family of such elements, which we refer to as Ta1 elements. Sequence analysis of one such element shows that it is closely related to retrotransposons and integrated retroviral proviruses, being bound by a direct sequence repeat and having an open reading frame with clear sequence similarity to the polyprotein of the Drosophila melanogaster retrotransposon copia. The sequence of an empty target site of a Ta1 element shows that insertion is accompanied by a five-base-pair target-site duplication and that Ta1 has transposed in the period of time since divergence of two races of A. thaliana.     

Role of transposable elements in heterochromatin and epigenetic control

Heterochromatin has been defined as deeply staining chromosomal material that remains condensed in interphase, whereas euchromatin undergoes de-condensation. Heterochromatin is found near centromeres and telomeres, but interstitial sites of heterochromatin (knobs) are common in plant genomes and were first described in maize. These regions are repetitive and late-replicating. In Drosophila, heterochromatin influences gene expression, a heterochromatin phenomenon called position effect variegation. Similarities between position effect variegation in Drosophila and gene silencing in maize mediated by "controlling elements" (that is, transposable elements) led in part to the proposal that heterochromatin is composed of transposable elements, and that such elements scattered throughout the genome might regulate development. Using microarray analysis, we show that heterochromatin in Arabidopsis is determined by transposable elements and related tandem repeats, under the control of the chromatin remodelling ATPase DDM1 (Decrease in DNA Methylation 1). Small interfering RNAs (siRNAs) correspond to these sequences, suggesting a role in guiding DDM1. We also show that transposable elements can regulate genes epigenetically, but only when inserted within or very close to them. This probably accounts for the regulation by DDM1 and the DNA methyltransferase MET1 of the euchromatic, imprinted gene FWA, as its promoter is provided by transposable-element-derived tandem repeats that are associated with siRNAs.     

Sequence of retrovirus provirus resembles that of bacterial transposable elements

The nucleotide sequences of the terminal regions of an infectious integrated retrovirus cloned in the modified lambda phage cloning vector Charon 4A have been elucidated. There is a 569-base pair direct repeat at both ends of the viral DNA. The cell-virus junctions at each end consist of a 5-base pair direct repeat of cell DNA next to a 3-base pair inverted repeat of viral DNA. This structure resembles that of a transposable element and is consistent with the protovirus hypothesis that retroviruses evolved from the cell genome.     

Hinf family: a novel repeated DNA family of the human genome

The isolation of a mutant adenovirus carrying an insertion of cellular DNA has led to the identification of a new family of human repetitive sequences, which are found tandemly arranged in the genome. The sequence of the viral insert resembles that of eukaryotic transposable elements.     

High-frequency precise excision of the Drosophila foldback transposable element

Precise excision of transposable elements in prokaryotes is a rare event which occurs at a significantly lower rate than transposition and other element-mediated events. Thus, we were intrigued by a eukaryotic transposable element which seemed capable of precise excision at high frequencies. The white-crimson (wc) mutation in Drosophila, a highly unstable allele of the X-linked eye colour locus, white, resulted from the insertion of a member of the foldback (FB) transposable element family. This mutation reverts to its parental phenotype at a frequency of greater than 1 in 10(3) X chromosomes. Characterization of these revertants by Southern blots of genomic DNA indicated that they resulted from loss of the wc insertion. Here we report the nucleotide sequence of the excision point in these revertants, and conclude that the FB element responsible for the wc mutation is capable of precise excision at high frequencies.     

Activation of a transposable element in the germ line but not the soma of Caenorhabditis elegans

The genetic activity of transposable elements is tightly controlled in many species. Transposons that are relatively quiescent under certain circumstances can excise or transpose at greatly increased rates under other circumstances. For example, 'genomic shock' can activate quiescent maize transposons, 'cytotype' and tissue-specific splicing regulate Drosophila P factors, copy number controls Tn5 transposition in bacteria, and developmental timing affects the production of transposon-like intracisternal A-particles in mouse embryos. The Caenorhabditis elegans transposable element Tc1 is subject to both strain-specific and tissue-specific control. Multiple copies of Tc1 are present in the genome of all C. elegans strains collected from nature. However, these elements are genetically active in only certain isolates. For example, in C. elegans variety Bristol transposition and excision of Tc1 are undetectable, but in variety Bergerac transposition and excision are frequent. Moreover, in variety Bergerac, Tc1 is about 1,000-fold more active in somatic cells than in germ cells. We have investigated the genetic basis for the germ/soma regulation of Tc1 activity. We have isolated mutants that exhibit increased frequencies of Tc1 excision in the germ line. The frequencies of Tc1 excision in the soma are unaltered in these mutants. These mutants also exhibit high frequencies of Tc1 germ-line transposition, and this results in a mutator phenotype. Nearly all mutator-induced mutations are caused by insertion of Tc1.     

短柄五加(Acanthopanax brachypus)rbcL基因的结构分析

克隆了含完整短柄五加rbcL基因的3.2kb EcoRI片段,测定了该基因的核苷酸序列.所测核苷酸序列总长度为1924bp,其中编码区1428bp,编码475个氨基酸的蛋白质.测定的基因5’上游区共278bp,包含原核性质-35区(TTGCGC),-10区(TACAAT)及类似真核的TATA box元件(TATATA).5’前导区长194bp,其中SD序列为GGAGG,紧邻起始密码子上游.测定的3’下游区共218bp,含2个相邻的转录后可形成茎环结构的反向重复序列.短柄五加rbcL基因编码区推导的氨基酸序列与烟草、菠菜、豌豆、苜蓿、玉米、水稻、松树、地钱、衣藻和Anacystis的同源性分别为93.5％、94.11％、94.53％、94.74％、89.68％、92.21％、92.21％、92.63％、87.58％和80.84％.本文还对不同植物rbcL基因的启动区及部分5’和3’非编码区进行了比较分析.     

A miniature insertion element transposable in Microcystis sp. FACHB 854

A 186-bp sequence with imperfect terminal inverted repeats and target direct repeats but without any transposase-encoding capacity was found to be transposable in an isolate derived from Microcystis sp. FACHB 854. This miniature insertion element, designated as ISM854-1, and with its homologues present at least 10 copies in the genome of Microcystis FACHB 854, is inserted into the 8-bp long and AT-rich target sequences, but none or few in other Microcystis strains. A variant of ISM854-1, denoted ISM854-1A, has perfect inverted repeat sequences and may transpose in pairs in a structure like a composite transposon. This is the first report of non-autonomous transposition of a mini-IS in a cyanobacterium.     

The plant MITE mPing is mobilized in anther culture

Transposable elements constitute a large portion of eukaryotic genomes and contribute to their evolution and diversification. Miniature inverted-repeat transposable elements (MITEs) constitute one of the main groups of transposable elements and are distributed ubiquitously in the genomes of plants and animals such as maize, rice, Arabidopsis, human, insect and nematode. Because active MITEs have not been identified, the transposition mechanism of MITEs and their accumulation in eukaryotic genomes remain poorly understood. Here we describe a new class of MITE, called miniature Ping (mPing), in the genome of Oryza sativa (rice). mPing elements are activated in cells derived from anther culture, where they are excised efficiently from original sites and reinserted into new loci. An mPing-associated Ping element, which has a putative PIF family transposase, is implicated in the recent proliferation of this MITE family in a subspecies of rice.     

Polymorphism near the rat prolactin gene caused by insertion of an Alu-like element

Primate Alu and rodent Alu-like elements comprise major families of mammalian small dispersed repetitive DNAs. These elements are repeated more than 10(5) times per haploid genome and are found between known genes, in introns and in satellite DNA. Their dispersion throughout the genome and the presence of directly repeated DNA sequences flanking the elements suggest, but do not prove, that they are capable of transposition. We describe here an allelic variation in the 5'-flanking region of the rat prolactin gene that offers the opportunity to examine the sequences of matching regions of two homologous chromosomes which differ in the presence of an Alu-like repetitive DNA element. Our findings support the hypothesis that these elements are integrated into the genome by generating short direct repeats of host DNA.     

Unusual sequences in the murine immunoglobulin mu-delta heavy-chain region

The delta heavy (H) chain of mouse immunoglobulin D (IgD) is unusual both in its structure and in its differential expression relative to immunoglobulin M (IgM; reviewed in ref. 1). The region of DNA between IgM and IgD H-chain constant-region genes is probably implicated in this control. So far only fragments of the area have been sequenced. Now, however, we present the complete sequence as well as the sequence of the introns of the C delta gene. We have found several interesting features (Fig. 1), including an open reading frame (ORF) between Cmu and C delta which encodes 146 amino acids that might represent a previously unsuspected domain-like protein; three blocks of simple repetitive sequences; a 162-base pair (bp) unique-sequence inverted repeat; and a domain-like pseudogene in the large intron of C delta. We have not found, however, any sequence 5' of C delta resembling the switch (S) recombination sequences associated with class switching in other heavy chains. Moreover, we have determined the 3' deletion end point of an IgD-producing myeloma and find no sequences reminiscent of switch sites nearby.     

A truncated human chromosome 16 associated with alpha thalassaemia is stabilized by addition of telomeric repeat (TTAGGG)n

The instability of chromosomes with breaks induced by X-irradiation led to the proposal that the natural ends of chromosomes are capped by a specialized structure, the telomere. Telomeres prevent end-to-end fusions and exonucleolytic degradation, enable the end of the linear DNA molecule to replicate, and function in cell division. Human telomeric DNA comprises approximately 2-20 kilobases (kb) of the tandemly repeated sequence (TTAGGG)n oriented 5'----3' in towards the end of the chromosome, interspersed with variant repeats in the proximal region. Immediately subtelomeric lie families of unrelated repeat motifs (telomere-associated sequences) whose function, if any, is unknown. In lower eukaryotes the formation and maintenance of telomeres may be mediated enzymatically (by telomerase) or by recombination; in man the mechanisms are poorly understood, although telomerase has been identified in HeLa cells. Here we describe an alpha thalassaemia mutation associated with terminal truncation of the short arm of chromosome 16 (within band 16p13-3) to a site 50 kb distal to the alpha globin genes, and show that (TTAGGG)n has been added directly to the site of the break. The mutation is stably inherited, proving that telomeric DNA alone is sufficient to stabilize the broken chromosome end. This mechanism may occur in any genetic disease associated with chromosome truncation.