Transformation of Neurospora crassa with recombinant plasmids containing the cloned glutamate dehydrogenase (am) gene: evidence for autonomous replication of the transforming plasmid.

We have characterized Neurospora crassa transformants obtained with plasmid pJR2, which consists of the Neurospora glutamate dehydrogenase (am) gene cloned in pUC8 and an am132 host strain which contains a deletion encompassing the cloned fragment. Every one of 33 transformants tested showed extreme meiotic instability: less than 1 or 2% am+ progeny were obtained in initial or successive backcrosses between am+ transformants and am132 or in intercrosses between am+ progeny. Furthermore, am+ progeny from backcrosses gave a high proportion of auxotrophic (am) mitotic segregants during vegetative growth. These results indicate that the am+ character is not stably integrated into chromosomal DNA in any of the transformants tested. Nuclear DNAs from six transformants were analyzed by Southern hybridization. All six transformants contained sequences homologous to pJR2. Four showed restriction fragments expected for unmodified pJR2, but most showed additional bands. Southern blots of undigested DNAs showed that the plasmid sequences are present predominantly in high-molecular-weight form (larger than 20 kilobases). Southern blots showed that auxotrophic (am) progeny from a backcross to am132 had lost restriction bands corresponding to free plasmid but retained additional bands, apparently integrated into chromosomal DNA in a nonfunctional manner. Considered together, these results are most reasonably interpreted as follows: recombinant plasmids containing the am+ gene can replicate autonomously in N. crassa, the free plasmids are present in oligomeric or modified form or both, and plasmid sequences also integrate at multiple sites in the deletion host but in a nonfunctional manner. An alternate interpretation--that tandem repeats of the plasmid are integrated into chromosomal DNA but eliminated during meiosis--cannot be completely excluded. However, stable integration of the am gene can be obtained under a variety of other conditions, viz., using the am gene cloned in a phage lambda vector (J. A. Kinsey and J. A. Rambosek, Mol. Cell. Biol. 4:117-122, 1984), using derivatives of pJR2, or using pJR2 to transform a frameshift mutant.     

Progressive rearrangement of telomeric sequences added to both the ITR ends of the yeast linear pGKL plasmid

Relocation into the nucleus of the yeast cytoplasmic linear plasmids was studied using a monitor plasmid pCLU1. InSaccharomyces cerevisiae, the nuclearly-relocated pCLU1 replicated in a linear form (termed pTLU-type plasmid) which carried the host telomeric repeats TG_1–3 of 300–350 bp at both ends. The telomere sequences mainly consisted of a major motif TGTGTGGGTGTGG which was complementary to part of the RNA template of yeast telomerase and were directly added to the very end of the pCLU1-terminal element ITR (inverted terminal repeat), suggesting that the ITR end played a role as a substrate of telomerase. The telomere sequences varied among isolated pTLU-type plasmids, but the TG_1–3 organization was symmetrically identical on both ends of any one plasmid. During cell growth under non-selective condition, the telomeric repeat sequences were progressively rearranged on one side, but not on the opposite side of pTLU plasmid ends. This indicates that the mode of telomeric DNA replication or repair differed between both ends. Clonal analysis showed that the intense rearrangement of telomeric DNA was closely associated with extreme instability of pTLU plasmids. Published: February 17, 2003     

Characterization of Chromosome Ends in the Filamentous Fungus Neurospora crassa

Telomeres and subtelomere regions have vital roles in cellular homeostasis and can facilitate niche adaptation. However, information on telomere/subtelomere structure is still limited to a small number of organisms. Prior to initiation of this project, the Neurospora crassa genome assembly contained only 3 of the 14 telomeres. The missing telomeres were identified through bioinformatic mining of raw sequence data from the genome project and from clones in new cosmid and plasmid libraries. Their chromosomal locations were assigned on the basis of paired-end read information and/or by RFLP mapping. One telomere is attached to the ribosomal repeat array. The remaining chromosome ends have atypical structures in that they lack distinct subtelomere domains or other sequence features that are associated with telomeres in other organisms. Many of the chromosome ends terminate in highly AT-rich sequences that appear to be products of repeat-induced point mutation, although most are not currently repeated sequences. Several chromosome termini in the standard Oak Ridge wild-type strain were compared to their counterparts in an exotic wild type, Mauriceville. This revealed that the sequences immediately adjacent to the telomeres are usually genome specific. Finally, despite the absence of many features typically found in the telomere regions of other organisms, the Neurospora chromosome termini still retain the dynamic nature that is characteristic of chromosome ends.     

Transformation of Aspergillus nidulans by the orotidine-5'-phosphate decarboxylase gene of Neurospora crassa

Relief of an auxotrophic requirement for uridine in Aspergillus nidulans strain G191 has been achieved by transformation with a segment of Neurospora crassa DNA containing the corresponding gene coding for orotidine-5'-phosphate decarboxylase. The mitotic stability of such transformants suggests that the DNA has integrated into the genome. Southern hybridisation analysis of DNA isolated from transformants revealed the presence of pBR322 sequences which have integrated into the host genome along with the N. crassa DNA.     

Transformation of Neurospora crassa by an integrative transforming plasmid is not enhanced by ribosomal DNA sequences

Two integrative transforming plasmids of Neurospora crassa that differed only by the presence of almost all of a ribosomal DNA repeat unit on one plasmid were constructed. The plasmids were used to test the target concentration hypothesis which states that the transformation frequency is proportional to the number of genomic copies of a homologous sequence located on the transforming plasmid. Since there are approx. 200 copies of the rDNA sequences in the genome, the target concentration hypothesis would have been proved if the transformation frequency was 200-fold higher for the rDNA-containing plasmid compared with the plasmid without rDNA. The results indicated no difference in the transformation for the two plasmids, thereby providing no support for the hypothesis. The target concentration hypothesis has been proved for yeast, and thus mechanisms different from that responsible for integrative transformation in yeast must operate in N. crassa, perhaps including non-homologous recombination events.     

Expression of minute virus of mice structural proteins in murine cell lines transformed by bovine papillomavirus-minute virus of mice plasmid chimera.

Recombinant plasmids containing the genomes of both bovine papillomavirus type I and minute virus of mice (MVM) were constructed and used to transform mouse C127 cells. Transformed lines that express MVM gene products with high efficiency were isolated and characterized. These transformants synthesize large amounts of MVM structural polypeptides and spontaneously assemble them into empty virion particles that are released into the culture medium. These lines were, however, genetically unstable; they slowly generated subpopulations that failed to express MVM-specific proteins, and they possessed episomal DNA in which both MVM and bovine papillomavirus sequences were deleted or rearranged, or both. Clonal isolates of these transformants were also superinfectible by infectious MVM virus. Therefore, in spite of their instability, they should be useful host cell lines for transcomplementing mutations introduced into the MVM genome and for growing defective viruses as virions.     

Analysis of DNA repeats in bacterial plasmids reveals the potential for recurrent instability events

Structural instability has been frequently observed in natural plasmids and vectors used for protein expression or DNA vaccine development. However, there is a lack of information concerning hotspot mapping, namely, DNA repeats or sequences identical to the host genome. This led us to evaluate the abundance and distribution of direct, inverted, and tandem repeats with high recombination potential in 36 natural plasmids from ten bacterial genera, as well as in several widely used bacterial and mammalian expression vectors. In natural plasmids, we observed an overrepresentation of close direct repeats in comparison to inverted ones and a preferential location of repeats with high recombination potential in intergenic regions, suggesting a highly plastic and dynamic behavior. In plasmid vectors, we found a high density of repeats within eukaryotic promoters and non-coding sequences. As a result of this in silico analysis, we detected a spontaneous recombination between two 21-bp direct repeats present in the human cytomegalovirus early enhancer/promoter (huCMV EEP) of the pCIneo plasmid. This finding is of particular importance, as the huCMV EEP is one of the most frequently used regulatory elements in plasmid vectors. Because pDNA integration into host gDNA can have adverse consequences in terms of plasmid processing and host safety, we also mapped several regions with high probability to mediate integration into the Escherichia coli or human genomes. Like repeated regions, some of these were located in non-coding regions of the plasmids, thus being preferential targets to be removed.     

The influence of interstitial telomeric sequences on chromosome instability in human cells

Although most telomere repeat sequences are found at the ends of chromosomes, some telomeric repeat sequences are also found at intrachromosomal locations in mammalian cells. Several studies have found that these interstitial telomeric repeat sequences can promote chromosome instability in rodent cells, either spontaneously or following ionizing radiation. In the present study we describe the extensive cytogenetic analysis of three different human cell lines with plasmids containing telomeric repeat sequences integrated at interstitial sites. In two of these cell lines, Q18 and P8SX, instability has been detected in the chromosome containing the integrated plasmid, involving breakage/fusion/bridge cycles or amplification of the plasmid DNA, respectively. However, the data suggest that the instability observed is characteristic of the general instability in these cell lines and that the telomeric repeat sequences themselves are not responsible. Consistent with this interpretation, the chromosome containing an integrated plasmid with 500 bp of telomeric repeat sequences is highly stable in the third cell line, SNG28, which has a relatively stable genome. The stability of the chromosome containing the integrated plasmid sequences in SNG28 makes this an excellent cell line to study the effect of ionizing radiation on the stability of interstitial telomeric sequences in human cells.     

Specificity of Repeat-Induced Point Mutation (Rip) in Neurospora: Sensitivity of Non-Neurospora Sequences, a Natural Diverged Tandem Duplication, and Unique DNA Adjacent to a Duplicated Region

The process designated RIP (repeat-induced point mutation) alters duplicated DNA sequences in the sexual cycle of Neurospora crassa. We tested whether non-Neurospora sequences are susceptible to RIP, explored the basis for the observed immunity to this process of a diverged tandem duplication that probably arose by a natural duplication followed by RIP (the Neurospora zeta-eta region), and investigated whether RIP extends at all into unique sequences bordering a duplicated region. Bacterial sequences of the plasmid pUC8 and of a gene conferring resistance to hygromycin B were sensitive to RIP in N. crassa when repeated in the genome. When the entire 1.6-kb zeta-eta region was duplicated, it was susceptible to RIP, but was affected by it to a lesser extent than other duplications. Only three of 62 progeny from crosses harboring unlinked duplications of the region showed evidence of changes. We attribute the low level of alterations to depletion of mutable sites. The stability of the zeta-eta region in strains having single copies of the region suggests that the 14% divergence of the tandem elements is sufficient to prevent RIP. DNA sequence analysis of unduplicated pUC8 sequences adjacent to a duplication revealed that RIP continued at least 180 bp beyond the boundary of the duplication. Three mutations occurred in the 200-bp segment of bordering sequences examined.     

An insertion sequence-dependent plasmid rearrangement in Aeromonas salmonicida causes the loss of the type three secretion system

Aeromonas salmonicida, a bacterial fish pathogen, possesses a functional Type Three Secretion System (TTSS), which is essential for its virulence. The genes for this system are mainly located in a single region of the large pAsa5 plasmid. Bacteria lose the TTSS region from this plasmid through rearrangements when grown in stressful growth conditions. The A. salmonicida genome is rich in insertion sequences (ISs), which are mobile DNA elements that can cause DNA rearrangements in other bacterial species. pAsa5 possesses numerous ISs. Three IS11s from the IS256 family encircle the rearranged regions. To confirm that these IS11s are involved in pAsa5 rearrangements, 26 strains derived from strain A449 and two Canadian isolates (01-B526 and 01-B516) with a pAsa5 rearrangement were tested using a PCR approach to determine whether the rearrangements were the result of an IS11-dependent process. Nine out of the 26 strains had a positive PCR result, suggesting that the rearrangement in these strains were IS-dependent. The PCR analysis showed that all the rearrangements in the A449-derived strains were IS11-dependent process while the rearrangements in 01-B526 and 01-B516 could only be partially coupled to the action of IS11. Unidentified elements that affect IS-dependent rearrangements may be present in 01-B526 and 01-B516. Our results suggested that pAsa5 rearrangements involve IS11. This is the first study showing that ISs are involved in plasmid instability in A. salmonicida.     

Fate of DNA encoding hygromycin resistance after meiosis in transformed strains of Gibberella fujikuroi (Fusarium moniliforme)

Stability of foreign DNA transformed into a novel host is an important parameter in decisions to permit the release of genetically engineered microorganisms into the environment. Meiotic instability of transformed DNA has been reported in fungi such as Ascobolus, Aspergillus, and Neurospora. We used strains of Gibberella fujikuroi (Fusarium moniliforme) transformed with the hygr gene from Escherichia coli to study meiotic stability of foreign DNA in this plant pathogenic fungus. Crosses with single-copy transformants segregated hygr:hygs in a 1:1 manner consistent with that expected for a Mendelian locus in a haploid organism. Multicopy transformants, however, segregated hygr:hygs in a 1:2 manner that was not consistent with Mendelian expectations for a chromosomal marker, even though two unrelated auxotrophic nuclear genes were segregating normally. Segregation ratios in crosses in which hygr was introduced via the male parent did not differ significantly from crosses in which the transformed strain served as the female parent. Some of the sensitive progeny from the crosses with the multicopy transformants carried hygr sequences. When these phenotypically sensitive progeny were crossed with a wild-type strain that carried no hygr sequences, some of the progeny were phenotypically hygr. Some progeny from some crosses were more resistant to hygromycin than were their sibs or the transformant strains that served as their parents. Transformants passaged through a maize plant only rarely segregated progeny with the high levels of resistance. The mechanism underlying these genetic instabilities is not clear but may involve unequal crossing over or methylation or both. Further work with cloned genes with homology to sequences already present in the Fusarium genome is warranted.     

Fate of DNA encoding hygromycin resistance after meiosis in transformed strains of Gibberella fujikuroi (Fusarium moniliforme).

Stability of foreign DNA transformed into a novel host is an important parameter in decisions to permit the release of genetically engineered microorganisms into the environment. Meiotic instability of transformed DNA has been reported in fungi such as Ascobolus, Aspergillus, and Neurospora. We used strains of Gibberella fujikuroi (Fusarium moniliforme) transformed with the hygr gene from Escherichia coli to study meiotic stability of foreign DNA in this plant pathogenic fungus. Crosses with single-copy transformants segregated hygr:hygs in a 1:1 manner consistent with that expected for a Mendelian locus in a haploid organism. Multicopy transformants, however, segregated hygr:hygs in a 1:2 manner that was not consistent with Mendelian expectations for a chromosomal marker, even though two unrelated auxotrophic nuclear genes were segregating normally. Segregation ratios in crosses in which hygr was introduced via the male parent did not differ significantly from crosses in which the transformed strain served as the female parent. Some of the sensitive progeny from the crosses with the multicopy transformants carried hygr sequences. When these phenotypically sensitive progeny were crossed with a wild-type strain that carried no hygr sequences, some of the progeny were phenotypically hygr. Some progeny from some crosses were more resistant to hygromycin than were their sibs or the transformant strains that served as their parents. Transformants passaged through a maize plant only rarely segregated progeny with the high levels of resistance. The mechanism underlying these genetic instabilities is not clear but may involve unequal crossing over or methylation or both. Further work with cloned genes with homology to sequences already present in the Fusarium genome is warranted.     

Sequence-specific epigenetic effects of the maternal somatic genome on developmental rearrangements of the zygotic genome in Paramecium primaurelia.

In ciliates, the germ line genome is extensively rearranged during the development of the somatic macronucleus from a mitotic product of the zygotic nucleus. Germ line chromosomes are fragmented in specific regions, and a large number of internal sequence elements are eliminated. It was previously shown that transformation of the vegetative macronucleus of Paramecium primaurelia with a plasmid containing a subtelomeric surface antigen gene can affect the processing of the homologous germ line genomic region during development of a new macronucleus in sexual progeny of transformed clones. The gene and telomere-proximal flanking sequences are deleted from the new macronuclear genome, although the germ line genome remains wild type. Here we show that plasmids containing nonoverlapping segments of the same genomic region are able to induce similar terminal deletions; the locations of deletion end points depend on the particular sequence used. Transformation of the maternal macronucleus with a sequence internal to a macronuclear chromosome also causes the occurrence of internal deletions between short direct repeats composed of alternating thymines and adenines. The epigenetic influence of maternal macronuclear sequences on developmental rearrangements of the zygotic genome thus appears to be both sequence specific and general, suggesting that this trans-nucleus effect is mediated by pairing of homologous sequences.     

Characterization of bacteriophage P1 library containing inserts of Drosophila DNA of 75–100 kilobase pairs

A multiple-hit bacteriophage P1 library containing DNA fragments from Drosophila melanogaster in the size range 75–100 kb was created and subjected to a preliminary evaluation for completeness, randomness, fidelity, and clone stability. This P1 library presently contains 3840 individual clones, or approximately two genome equivalents. The library was screened with a small set of unique-sequence test probes, and clones containing the sequences have been recovered. In situ hybridization with salivary gland chromosomes indicates that the clones originate from the site of the probe sequences in the genome, and filter hybridization of restriction digests suggests that the clones are not rearranged in comparison with the genomic sequences. Approximately 1.7% of the clones contain sequences that hybridize with ribosomal DNA. A small subset of these clones was tested for stability by examination of restriction fragments produced after repeated subculturing, and no evidence for instability was found. The P1 cloning system has general utility in molecular genetics and may provide an important intermediate level of resolution in physical mapping of the Drosophila genome.by W. Hennig     

DNA疫苗安全性的策略探究

DNA疫苗安全性问题主要来自DNA疫苗元件和疫苗工程菌,包括DNA疫苗与宿主基因组整合、接种后诱导宿主产生免疫耐受和自身免疫疾病、抗性基因的转移问题、疫苗中内毒素和抗生素及其他有害物质残留问题、宿主菌体内DNA复制与纯化过程中基因稳定性问题。本文针对这些问题进行策略上的探讨,为进一步开发安全的DNA疫苗提供帮助。     

Deciphering the model pathogenic fungus Cryptococcus neoformans

Cryptococcus neoformans is a basidiomycete fungal pathogen of humans that has diverged considerably from other model fungi such as Neurospora crassa, Aspergillus nidulans, Saccharomyces cerevisiae and the common human fungal pathogen Candida albicans. The recent completion of the genome sequences of two related C. neoformans strains and the ongoing genome sequencing of three other divergent Cryptococcus strains with different virulence phenotypes and environmental distributions should improve our understanding of this important pathogen. We discuss the biology of C. neoformans in light of this genomic data, with a special emphasis on the role that evolution and sexual reproduction have in the complex relationships of the fungus with the environment and the host.     

Characterization of transgene integration pattern in F4 hGH-transgenic common carp （Cyprinus carpio L.）

The integration pattern and adjacent host sequences of the inserted pMThGH-transgene in the F4 hGH-transgenic common carp were extensively studied. Here we show that each F4 transgenic fish contained about 200 copies of the pMThGH-transgene and the transgenes were integrated into the host genome generally with concatemers in a head-totail arrangement at 4-5 insertion sites. By using a method of plasmid rescue, four hundred copies of transgenes from two individuals of F4 transgenic fish, A and B, were recovered and clarified into 6 classes. All classes of recovered transgenes contained either complete or partial pMThGH sequences. The class I, which comprised 83% and 84.5% respectively of the recovered transgene copies from fish A and B, had maintained the original configuration, indicating that most transgenes were faithfully inherited during the four generations of reproduction. The other five classes were different from the original configuration in both molecular weight and restriction map, indicating that a few transgenes had undergone mutation, rearrangement or deletion during integration and germline transmission. In the five types of aberrant transgenes, three flanking sequences of the host genome were analyzed. These sequences were common carp β-actin gene, common carp DNA sequences homologous to mouse phosphoglycerate kinase-1 and human epidermal keratin 14, respectively.     

Acquisition of telomere repeat sequences by transfected DNA integrated at the site of a chromosome break.

Previous analysis of plasmid DNA transfected into 108 cell clones demonstrated extensive polymorphism near the integration site in one clone. This polymorphism was apparent by Southern blot analysis as diffuse bands that extended over 30 kb. In the present study, nucleotide sequence analysis of cloned DNA from the integration site revealed telomere repeat sequences at the ends of the integrated plasmid DNA. The telomere repeat sequences at one end were located at the junction between the plasmid and cell DNA. The telomere repeat sequences at the other end were located in the opposite orientation in the polymorphic region and were shown by digestion with BAL 31 to be at the end of the chromosome. Telomere repeat sequences were not found at this location in the plasmid or parent cell DNA. Although the repeat sequences may have been acquired by recombination, a more likely explanation is that they were added to the ends of the plasmid by telomerase before integration. Comparison of the cell DNA before and after integration revealed that a chromosome break had occurred at the integration site, which was shown by fluorescent in situ hybridization to be located near the telomere of chromosome 13. These results demonstrate that chromosome breakage and rearrangement can result in interstitial telomere repeat sequences within the human genome. These sequences could promote genomic instability, because short repeat sequences can be recombinational hotspots. The results also show that DNA rearrangements involving telomere repeat sequences can be associated with chromosome breaks. The introduction of telomere repeat sequences at spontaneous or ionizing radiation-induced DNA strand breaks may therefore also be a mechanism of chromosome fragmentation.     

Recombination between irradiated shuttle vector DNA and chromosomal DNA in African green monkey kidney cells.

An autonomously replicating shuttle vector was used to investigate enhancement of plasmid-chromosome recombination in mammalian host cells by gamma irradiation and UV light. Sequences homologous to the shuttle vector were stably inserted into the genome of African green monkey kidney cells to act as the target substrate for these recombination events. The shuttle vector molecules were irradiated at various doses before transfection into the mammalian host cells that contained the stable insertions. The homologous transfer of the bacterial ampicillin resistance gene from the inserted sequences to replace a mutant ampicillin sensitivity gene on the shuttle vector was identified by the recovery of ampicillin-resistant plasmids after Hirt extraction and transformation into Escherichia coli host cells. Gamma irradiation increased homologous shuttle vector-chromosome recombination, whereas UV light did not increase the frequency of recombinant plasmids detected. Introducing specific double-strand breaks in the plasmid or prolonging the time of plasmid residence in the mammalian host cells also enhanced plasmid-chromosome recombination. In contrast, plasmid mutagenesis was increased by UV irradiation of the plasmid but did not change with time. The ampicillin-resistant recombinant plasmid molecules analyzed appeared to rise mostly from nonconservative exchanges that involved both homologous and possibly nonhomologous interactions with the host chromosome. The observation that similar recombinant structures were obtained from all the plasmid treatments and host cells used suggests a common mechanism for plasmid-chromosome recombination in these mammalian cells.