A cloned fragment of HeLa DNA containing consensus sequences of satellite II and III DNA hybridizes with the Drosophila P-element and with the 1.8 kb family of human KpnI fragments

We have cloned a repetitive EcoRI fragment from the human genome which displays weak homologies with the Drosophila melanogaster transposable P-element. This cloned DNA appeared not to be a mobile element but, instead, a divergent member of human satellite II or III DNAs. We present here the first complete nucleotide sequence of a 1.797 kilobase pair (kb) satellite-like DNA. Moreover, this EcoRI satellite monomer contains a unique sequence of 49 basepairs (bp) that is devoid of the satellite consensus repeat 5'TTCCA3'. Southern hybridization analysis revealed that the cloned insert is closely related to a highly repetitive 1.8 kb KpnI family of tandemly organized satellite DNAs. Thus, the relationships among these satellite DNA families appear to be complex and may be a factor in their copy number, position and spatial organization.     

A Schizosaccharomyces pombe artificial chromosome large DNA cloning system.

The feasibility of using the fission yeast, Schizosaccharomyces pombe , as a host for the propagation of cloned large fragments of human DNA has been investigated. Two acentric vector arms were utilized; these carry autonomously replicating sequences ( ars elements), selectable markers ( ura4(+) or LEU2 ) and 250 bp of S. pombe terminal telomeric repeats. All cloning was performed between the unique sites in both vector arms for the restriction endonuclease Not I. Initially the system was tested by converting six previously characterized cosmids from human chromosome 11p13 into a form that could be propagated in S.pombe as linear episomal elements of 50-60 kb in length. In all transformants analysed these cosmids were maintained intact. To test if larger fragments of human DNA could also be propagated total human DNA was digested with Not I and size fractionated by pulsed field gel electrophoresis (PFGE). Fractions of 100-1000 kb were ligated to Not I-digested vector arms and transformed into S.pombe protoplasts in the presence of lipofectin. Prototrophic ura+leu+transformants were obtained which upon examination by PFGE were found to contain additional linear chromosomes migrating at between 100 and 500 kb with a copy number of 5-10 copies/cell. Hybridization analyses revealed that these additional bands contained human DNA. Fluorescent in situ hybridization (FISH) analyses of several independent clones indicated that the inserts were derived from single loci within the human genome. These analyses clearly demonstrate that it is possible to clone large fragments of heterologous DNA in fission yeast using this S.p ombe artificial chromosome system which we have called SPARC. This vector-host system will complement the various other systems for cloning large DNA fragments.     

小葱植物内生链霉菌线型质粒pYY8L的端粒与复制区的克隆和鉴定

从小葱植物中分离到一株编号为36R-2-1B的链霉菌菌株,该菌株含有一个约为280kb的线型质粒pYY8L。【目的】克隆、测序和分析pYY8L新的端粒和复制区。【方法】采用改良的"在凝胶中进行DNA碱处理与酶切"的方法来克隆大的线型质粒pYY8L的端粒,通过构建基因组柯斯文库和次级克隆的方法来缩小和鉴定pYY8L的复制区。【结果】在小葱植物内生链霉菌36R-2-1B中检测到约为280kb的线型质粒pYY8L,克隆了pYY8L的端粒。其末端的152bp包含6个小的回文序列,可以形成复杂的二级结构。利用柯斯文库构建、次级克隆和测序获得了4891bp的pYY8L的复制区。该复制区含有6个基因,其中2个与天蓝色链霉菌线型质粒SCP1的复制基因非常相似,但是邻近的重复序列不同。【结论】采用新的改良的方法克隆和鉴定了pYY8L新的端粒和复制区。本文首次报道了植物内生链霉菌线型质粒的端粒和复制基因。     

Resistance to antimycin A in yeast by amplification of ADH4 on a linear, 42 kb palindromic plasmid

A yeast strain lacking a functional copy of ADH1 has been isolated that is resistant to antimycin A because of the presence of multiple copies of a nuclear gene, ADH4. The amplified copies of ADH4 exist on linear molecules 42 kb in length, which can be separated from chromosomal DNA by orthogonal-field-alternation gel electrophoresis. These amplified molecules are palindromes that reanneal rapidly after denaturation to form linear, snap-back molecules 21 kb in length. The amplified ADH4 sequences are bounded by telomere-homologous sequences. The chromosomal copy of ADH4 is the most distal marker on the left arm of chromosome VII, and the amplified ADH4-containing molecules appear to contain two copies of the region extending from ADH4 to the telomere.     

Efficient combination of large DNA in vitro: in gel site specific recombination (IGSSR) of PAC fragments containing alpha satellite DNA and the human HPRT gene locus.

In an attempt to combine a cloned genomic copy of a selectable gene with different cloned centromeric sequences to develop mammalian artificial chromosomes (MAC) we used site specific recombination mediated by purified Cre recombinase acting on the loxP sequence in PAC vector DNA. A new method was required to purify highly concentrated, virtually 100% intact PAC DNA which could be stored for a long period. Here we show the efficient linking of linearized PACs containing alpha satellite DNA from chromosomes X and 17 with sizes of 125 and 140 kb, respectively, to a 95 kb restriction fragment derived from a 175 kb PAC containing the intact human HPRT gene locus.     

A novel method for making nested deletions and its application for sequencing of a 300 kb region of human APP locus.

We developed a novel in vitro method for making nested deletions and applied it to a large-scale DNA sequencing. A DNA fragment to be sequenced (up to 15 kb long) was cloned with a new vector possessing two unique Sfi I sites, digested by Sfi I and ligated to generate a large head-to-tail concatemer. The large concatemer was randomly fragmented by sonication and then redigested by Sfi I to separate insert and vector DNAs. The fragments of various length were then cloned into the other vector(s) specifically designed for selective cloning of insert-derived DNA fragments to generate a library of nested deletions. This method allowed a single person to generate >20 nested deletion libraries sufficient to cover 100 kb in a few days. We applied the method for sequencing of P1 clones and successfully determined the complete sequence of approximately 300 kb of the human amyloid precursor protein (APP) locus on chromosome 21 with a redundancy of 3.8, reasonably low cost and very few gaps remaining to be closed. Development of some new instruments and software is also described which makes this method more applicable for large-scale sequencing.     

Organization of Plasmodium falciparum genome: 1. Evidence for a highly repeated DNA sequence.

Plasmodium falciparum DNA, isolated from the merozoite stage, was cleaved with HindIII and cloned in pBR322 and lambda L47.1 vectors. Plasmid clones containing 13.4, 7.0, 4.3, 4.1 and 1.5 kb inserts were characterized in some detail. The inserts contain several repeating units of smaller size. Nucleic acid hybridization studies showed that the repeat element is present in the Plasmodium DNA at a very high copy number and appears to be distributed widely throughout the genome.     

The 2-micron plasmid as a nonselectable, stable, high copy number yeast vector

The endogenous 2-microns plasmid of Saccharomyces cerevisiae has been used extensively for the construction of yeast cloning and expression plasmids because it is a native yeast plasmid that is able to be maintained stably in cells at high copy number. Almost invariably, these plasmid constructs, containing some or all 2-microns sequences, exhibit copy number levels lower than 2-microns and are maintained stably only under selective conditions. We were interested in determining if there was a means by which 2-microns could be utilized for vector construction, without forfeiting either copy number or nonselective stability. We identified sites in the 2-microns plasmid that could be used for the insertion of genetic sequences without disrupting 2-microns coding elements and then assessed subsequent plasmid constructs for stability and copy number in vivo. We demonstrate the utility of a previously described 2-microns recombination chimera, pBH-2L, for the manipulation and transformation of 2-microns as a pure yeast plasmid vector. We show that the HpaI site near the STB element in the 2-microns plasmid can be utilized to clone yeast DNA of at least 3.9 kb with no loss of plasmid stability. Additionally, the copy number of these constructs is as high as levels reported for the endogenous 2-microns.     

Intrastrand annealing leads to the formation of a large DNA palindrome and determines the boundaries of genomic amplification in human cancer

Amplification of large chromosomal regions (gene amplification) is a common somatic alteration in human cancer cells and often is associated with advanced disease. A critical event initiating gene amplification is a DNA double-strand break (DSB), which is immediately followed by the formation of a large DNA palindrome. Large DNA palindromes are frequent and nonrandomly distributed in the genomes of cancer cells and facilitate a further increase in copy number. Although the importance of the formation of large DNA palindromes as a very early event in gene amplification is widely recognized, it is not known how a DSB is resolved to form a large DNA palindrome and whether any local DNA structure determines the location of large DNA palindromes. We show here that intrastrand annealing following a DNA double-strand break leads to the formation of large DNA palindromes and that DNA inverted repeats in the genome determine the efficiency of this event. Furthermore, in human Colo320DM cancer cells, a DNA inverted repeat in the genome marks the border between amplified and nonamplified DNA. Therefore, an early step of gene amplification is a regulated process that is facilitated by DNA inverted repeats in the genome.     

Molecular analysis of DNA junctions produced by illegitimate recombination in human cells.

In a human HeLa derived-cell line carrying permanently a single integrated copy of an SV40 shuttle vector, the transient expression of the SV40 T-antigen led to the production of heterogeneous populations of circular DNA molecules which retained both integrated vector and its surrounding cellular sequences. Comparison between the integrated copy and the linear maps of 80 different plasmids rescued in bacteria suggested that the formation of circular DNA was the result of bidirectional replication from the SV40 origin of replication followed by a single intramolecular joining leading to the cyclization of the replicated molecules. Sequence analysis of 45 recombinational junctions demonstrated that the cyclization occurred via illegitimate recombination process which did not require preferential nucleotide sequence at the joining sites. However, extensive characterization of recombination junctions revealed that the sequences involved in the recombination at each side of the SV40 origin of replication were not randomly distributed, suggesting the presence of regions which were more prone to be involved in the illegitimate recombination process in human cells. Search of common features usually implied in illegitimate recombination in mammalian cells revealed some association of these regions with palindromes, A + T-rich DNA segments, alternating purine/pyrimidine sequences and Alu family repeats.     

Structure and distribution of inverted repeats (palindromes)

The size and distribution of renatured inverted repeats (palindromes) in D. melanogaster DNA were studied by electron microscopy (EM). The results of these studies differ from the previously published observations regarding the number, distribution and the size of inverted repeats (ir) present in DNA. -1. In contrast to the previous published observation almost all (96%) of the ir were found in crowded clusters. The DNA strands with clustered palindromes contained 2-21 palindromes (4-42 ir), with an average of 7.25 palindromes (14.5 ir) per strand. No correlation could be found between the length of the DNA strands and the number of ir per strand. -2, Also contrary to some previously published results, most (80%) of the ir formed on renaturation unlooped palindromes and these were always clustered. Looped palindromes (hairpins, formed by renaturation of ir separated by a non-homologous sequence long enough to be seen in EM as single-stranded loop) were found 1-2 per DNA strand, as part of clusters or as solitary palindromes in a DNA strand. The average spacing length (inside clusters) between centers of all palindromes was 2.349 kb, and between centers of looped palindromes 7.6 kb. - 3. The length of the ir was found to be smaller than documented in most of the previously published results. The majority, 80-90%, of the ir found in the unlooped and looped palindromes, respectively, belonged to one main-size class with a range of 30-210 bp and an average length of 100 bp, but longer ir were also observed. The average length of the ir in unlooped palindromes was 124 bp, in looped 244 bp, and the total average was 148 bp - 4. It was calculated that there are about 30,000 palindromes (60,000 ir) in the D, melanogaster genome, of which about 24,000 are unlooped and 6,000 looped, with the spacing between centers of all palindromes averaging about 4.4 kb in length.     

The structure of the human CD2 gene and its expression in transgenic mice.

We report the genomic organization of the human CD2 gene and its expression in transgenic mice. A 28.5 kb segment of DNA consisting of 4.5 kb 5' flanking sequences, 15 kb containing the gene's five exons and 9 kb of 3' flanking sequences can direct the expression of the CD2 gene only on thymocytes, circulating T cells and megakaryocytes of the transgenic mice. The expression of each copy of the human CD2 transgene appears to be as high as the endogenous mouse CD2 gene and as high as the expression on the surface of human T lymphocytes, independent of the site of integration and dependent on the copy number of genes that have integrated.     

Extrachromosomal replication of shuttle vectors in Dictyostelium discoideum.

We cloned a 12.3-kilobase (kb) endogenous plasmid, Ddp1, found in several wild-type and laboratory strains of Dictyostelium discoideum into pBR322. The cloned plasmids have been used to cotransform D. discoideum cells with B10S, a transformation vector carrying a gene fusion conferring resistance to G418. Whereas B10S DNA alone appears to integrate in a tandem array, the cloned Ddp1 plasmids replicate extrachromosomally and are stably maintained in the absence of selection with an average copy number of 50 to 100 copies per cell. The Ddp1-derived plasmids can be directly recovered by transforming Escherichia coli with bulk nuclear DNA from these cells. Preliminary deletion analysis indicates that not all regions of Ddp1 are necessary for stable replication in D. discoideum. Several recombinant vectors which replicate extrachromosomally in D. discoideum were also isolated. One contains the Act6-neor gene fusion from B10S recombined into one of the cloned derivatives of Ddp1 and can be used to directly transform D. discoideum amoebae, selecting for G418 resistance. Another recombinant is only 5.6 kb and resulted from a deletion of a 16.6-kb cloned Ddp1 hybrid plasmid. An analysis of the vector DNAs present in clones derived from single D. discoideum transformants is also described.     

Autonomous replication and addition of telomerelike sequences to DNA microinjected into Paramecium tetraurelia macronuclei.

Paramecium tetraurelia can be transformed by microinjection of cloned serotype A gene sequences into the macronucleus. Transformants are detected by their ability to express serotype A surface antigen from the injected templates. After injection, the DNA is converted from a supercoiled form to a linear form by cleavage at nonrandom sites. The linear form appears to replicate autonomously as a unit-length molecule and is present in transformants at high copy number. The injected DNA is further processed by the addition of paramecium-type telomeric sequences to the termini of the linear DNA. To examine the fate of injected linear DNA molecules, plasmid pSA14SB DNA containing the A gene was cleaved into two linear pieces, a 14-kilobase (kb) piece containing the A gene and flanking sequences and a 2.2-kb piece consisting of the procaryotic vector. In transformants expressing the A gene, we observed that two linear DNA species were present which correspond to the two species injected. Both species had Paramecium telomerelike sequences added to their termini. For the 2.2-kb DNA, we show that the site of addition of the telomerelike sequences is directly at one terminus and within one nucleotide of the other terminus. These results indicate that injected procaryotic DNA is capable of autonomous replication in Paramecium macronuclei and that telomeric addition in the macronucleus does not require specific recognition sequences.     

Microcloning of maize chromosome 9 by using a flow-sorting technique

We constructed a chromosome 9 lambda DNA library from flow-sorted maize chromosomes. Approximately 3 million maize chromosome 9 were collected with high purity by flow cytometric sorting of chromosomes isolated from an oat-maize chromosome 9 addition line based on the cytogram of fluorescent pulse area versus fluorescent pulse width. Chromosome 9 DNA was partially digested withBamH I, dephosphorylated, and ligated with arms ofBamH I-digested lambda DASH vector (Stratagene). A total of 2.0×10⁶ independent recombinants with an average insert size of 15 kb were obtained. For a 99% probability that every sequence of chromosome 9 is represented in at least one chimeric phage, 5.6×10⁴ cloned fragments are needed. This library covers the entire maize chromosome 9. Hybridizing cloned fragments with labeled maize genomic DNA showed that the high, middle, or low copy number DNA sequences presented in the different phage clones. This individual chromosome library is useful in plant genome mapping and gene isolation.     

Construction of an extrachromosomally replicating transformation vector for Dictyostelium discoideum

An extrachromosomally replicating transformation vector for Dictyostelium discoideum has been constructed using sequences of the endogenous Dictyostelium plasmid Ddp2. This transformation vector pnDeI (9.6 kb) replicates as a high copy number plasmid in Dictyostelium and is located in the nucleus. It has been constructed as shuttle vector containing the Escherichia coli vector pUC19 for replication and selection in E. coli and a part of the Tn903 transposon which confers resistance to G418 for selection in Dictyostelium. In order to show that the vector can be used for cloning and stable propagation of Dictyostelium DNA, a fragment of the Dictyostelium alpha-actinin gene that was marked with a synthetic oligonucleotide was cloned into pnDeI and found to be stably maintained in the extrachromosomal vector without undergoing noticeable recombination with the endogenous gene.     

Conjugational transfer system to shuttle giant DNA cloned by Bacillus subtilis genome (BGM) vector

The Bacillus subtilis GenoMe (BGM) vector was designed as a versatile vector for the cloning of giant DNA segments. Cloned DNA in the BGM can be retrieved to a plasmid using our Bacillus recombinational transfer (BReT) method that takes advantage of competent cell transformation. However, delivery of the plasmid to a different B. subtilis strain by the normal transformation method is hampered by DNA size-related inefficiency. Therefore, we designed a novel method, conjugational plasmid-mediated DNA retrieval and transfer (CReT) from the BGM vector, and investigated conjugational transmission to traverse DNA between cells to circumvent the transformation-induced size limitation. pLS20, a 65-kb plasmid capable of conjugational transfer between B. subtilis strains, was modified to retrieve DNA cloned in the BGM vector by homologous recombination during normal culture. As the plasmid copy number was estimated to be 3, the retrieval plasmid was selected using increased numbers of marker genes derived from the retrieved DNA. We applied this method to retrieve Synechocystis genome segments up to 90 kb in length. We observed retrieved plasmid transfers between B. subtilis strains by conjugation in the absence of structural alterations in the DNA fragment. Our observations extend DNA transfer protocols over previously exploited size ranges.     

Structure and distribution of inverted repeats (palindromes)

The size and distribution of renatured inverted repeats (palindromes) in Mus musculus DNA were examined by electron microscopy (EM). The majority (85%) of palindromes were found to be clustered in about one half of the DNA strands. The rest of the DNA strands were seen with a solitary looped structure. — The unlooped palindromes constituted 53% of all palindromes and were always clustered. There was a significant reduction in the number of unlooped palindromes in comparison to D. melanogaster DNA (Biezunski, 1981) and as a result the palindrome clusters were smaller and contained 2–8 palindromes [4–16 inverted repeats (ir)] per DNA strand. The looped palindromes had a wide and regular distribution with spacing lengths similar to those found in D. melanogaster DNA, and showed some periodicity. The average spacing between centers of all palindromes (inside a cluster) was 4.325 kb, and between centers of looped palindromes 8.544 kb. — The lengths of the ir of unlooped and looped palindromes were grouped (similar to D. melanogaster DNA) in one size-class with a range of 30–240 bp and an average length of 130 bp. Longer ir were also observed and the average length of ir in unlooped palindromes was 186 bp, in looped 588 bp, and the total average length was 375 bp. — It was calculated that there are about 224,000–320,000 palindromes (ir pairs) in the mouse genome, with the spacing between centers of all palindromes about 13-9 kb in length. — In high molecular weight mouse DNA, complex looped structures composed of rows of 5–8 looped palindromes one on top the other, formed by renaturation of multiple ir, were observed. It is suggested, that clustered repetitive sequences, in direct and inverted orientation, might be of one family and homologous to one another, and be able to reassociate, in vitro and in vivo, into structures of different forms, which could function as binding sites for various regulatory proteins during mouse development.