Recipient Chromosome Research Articles

Horizontal Transfer of the High-Pathogenicity Island ofYersinia pseudotuberculosis

The horizontal transfer of genetic elements plays a major role in bacterial evolution. The high-pathogenicity island (HPI), which codes for an iron uptake system, is present and highly conserved in various Enterobacteriaceae, suggesting its recent acquisition by lateral gene transfer. The aim of this work was to determine whether the HPI has kept its ability to be transmitted horizontally. We demonstrate here that the HPI is indeed transferable from a donor to a recipient Yersinia pseudotuberculosis strain. This transfer was observable only when the donor and recipient bacteria were cocultured at low temperatures in a liquid medium. When optimized conditions were used (bacteria actively growing in an iron-deprived medium at 4 degrees C), the frequency of HPI transfer reached approximately 10(-8). The island was transferable to various serotype I strains of Y. pseudotuberculosis and to Yersinia pestis, but not to Y. pseudotuberculosis strains of serotypes II and IV or to Yersinia enterocolitica. Upon transfer, the HPI was inserted almost systematically into the asn3 tRNA locus. Acquisition of the HPI resulted in the loss of the resident island, suggesting an incompatibility between two copies of the HPI within the same strain. Transfer of the island did not require a functional HPI-borne insertion-excision machinery and was RecA dependent in the recipient but not the donor strain, suggesting that integration of the island into the recipient chromosome occurs via a mechanism of homologous recombination. This lateral transfer also involved the HPI-adjacent sequences, leading to the mobilization of a chromosomal region at least 46 kb in size.

A Salmonella typhimurium Effector Protein SifA Is Modified by Host Cell Prenylation and S-Acylation Machinery

SifA is a Salmonella effector protein that is required for maintenance of the vacuolar membrane that surrounds replicating bacteria. It associates with the Salmonella-containing vacuole but how it interacts with the membrane is unknown. Here we show by immunofluorescence, S100 fractionation and Triton X-114 partitioning that the membrane association and targeting properties of SifA are influenced by a motif encoded within the C-terminal six amino acids. This sequence shares homology with both CAAX and Rab geranylgeranyl transferase prenylation motifs. We characterized the post-translational processing of SifA and showed that the cysteine residue within the CAAX motif is modified by isoprenoid addition through the action of protein geranylgeranyl transferase I. SifA was additionally modified by S-acylation of an adjacent cysteine residue. Similar modifications to host cell proteins regulate numerous functions including protein targeting, membrane association, protein-protein interaction, and signal transduction. This is the only known example of a bacterial effector protein that is modified both by mammalian cell S-acylation and prenylation machinery.

The Salmonella genomic island 1 is an integrative mobilizable element

Salmonella genomic island 1 (SGI1) is a genomic island containing an antibiotic resistance gene cluster identified in several Salmonella enterica serovars. The SGI1 antibiotic resistance gene cluster, which is a complex class 1 integron, confers the common multidrug resistance phenotype of epidemic S. enterica Typhimurium DT104. The SGI1 occurrence in S. enterica serovars Typhimurium, Agona, Paratyphi B, Albany, Meleagridis and Newport indicates the horizontal transfer potential of SGI1. Here, we report that SGI1 could be conjugally transferred from S. enterica donor strains to non-SGI1 S. enterica and Escherichia coli recipient strains where it integrated into the recipient chromosome in a site-specific manner. First, an extrachromosomal circular form of SGI1 was identified by PCR which forms through a specific recombination of the left and right ends of the integrated SGI1. Chromosomal excision of SGI1 was found to require SGI1-encoded integrase which presents similarities to the lambdoid integrase family. Second, the conjugal transfer of SGI1 required the presence of a helper plasmid. The conjugative IncC plasmid R55 could thus mobilize in trans SGI1 which was transferred from the donor to the recipient strains. By this way, the conjugal transfer of SGI1 occurred at a frequency of 10(-5)-10(-6) transconjugants per donor. No transconjugants could be obtained for the SGI1 donor lacking the int integrase gene. Third, chromosomal integration of SGI1 occurred via a site-specific recombination between a 18 bp sequence found in the circular form of SGI1 and a similar 18 bp sequence at the 3' end of thdF gene in the S. enterica and E. coli chromosome. SGI1 appeared to be transmissible only in the presence of additional conjugative functions provided in trans. SGI1 can thus be classified within the group of integrative mobilizable elements (IMEs).

Plasmid DNA transfer in Mycobacterium smegmatis involves novel DNA rearrangements in the recipient, which can be exploited for molecular genetic studies.

The establishment of molecular genetic techniques is essential for development of new treatments for mycobacterial infections. To this end, we recently described a novel DNA transfer process that occurs in the model mycobacterial organism Mycobacterium smegmatis. This transfer system is most like conjugal DNA transfer in that it requires two viable parents, is DNAse resistant and occurs between distinct donor and recipient strains. Cis-acting sequences called bom, which confer transferability, are distinct from the prototypical oriT sites of conjugative plasmids, as they occur at multiple locations in the chromosome and require RecA in the recipient to mediate plasmid recircularization. Here, we show that a plasmid containing two of these bom regions can undergo several fates in the recipient cell, each of which require recipient recombination functions. The products of plasmid transfer that we observed provide further insights toward a model for DNA transfer. Furthermore, we have taken advantage of the recombination events that occur in the recipient to develop simple procedures for capturing, or replacing specific segments of the recipient chromosome. To demonstrate the potential of the system, we describe the capture and deletion of 25 kb of the M. smegmatis chromosome, and targeted-allele exchange of the recipient recB and recD genes. Using these transfer-mediated rearrangements, we demonstrate that homology with the recipient chromosome and RecB, but not RecD, are essential for DNA transfer.

Intracellular expression of Vitreoscilla hemoglobin in Aspergillus terreus to alleviate the effect of a short break in aeration during culture.

A continuous supply of O(2) is important for itaconic acid production in Aspergillus terreus. Any interruption of aeration significantly reduces itaconic acid production. To overcome this effect, A. terreus M8 was transformed with the Vitreoscilla hemoglobin gene (vgb) which, as shown by Southern hybridization, was integrated into the recipient chromosome. The activity of the expressed hemoglobin was confirmed by a CO-difference spectrum. During itaconic acid production, the effect of a break in aeration during cultivation in the transformant with the vgb gene is alleviated. Additionally, the transformant shows improved itaconic acid production.

Genetic transformation and mutant isolation in Ganoderma lucidum by restriction enzyme-mediated integration

A white-rot basidiomycete Ganoderma lucidum has long been used as a medicinal mushroom in Asia, and it has an array of enzymes important for wood degrading activity. There have been many reports about the ingredients which show health aiding effects. In order to analyze gene functions and introduce foreign genes into this fungus, genetic transformation is required. We have successfully transformed G. lucidum to geneticin resistance using pJS205-1 which has the antibiotic resistance genes against geneticin and phosphinothricin. Many different mutants have been generated during the transformation by restriction enzyme mediated integration, and the transformation yield was 4–17 transformants (μg plasmid DNA) −1. The plasmid was integrated stably into the recipient chromosome, which was confirmed by PCR with the plasmid-specific primers.

Evaluation of host stem cell–derived cardiac myocytes in consecutive biopsies in long-term cardiac transplant patients

Background Bone marrow–derived stem cells are involved in tissue formation in transplanted organs. In human renal cell transplants, recipient-specific cells have been shown to participate in regeneration of interstitial tissue. In hepatic transplants, hepatocytes with a recipient sex chromosome pattern have been observed. Recruitment of stem cells in repair and regeneration of cardiac myocytes has been demonstrated in experimental ischemia. Recently, cardiac antral myocytes of recipient origin have been shown to populate transplanted hearts in gender-mismatched transplants. Methods Using the flourescence in situ hybridization (FISH) technique we examined serial post-transplant right ventricular biopsies and sections of ventricle (at time of autopsy) of 4 gender-mismatched female-to-male transplanted hearts (graft survival 0.5 to 10 years, mean 5.1 years) for Y chromosomes within cardiac myocytes. Results We detected recipient-specific sex chromosomal patterns in single rare cardiac myocytes in 1 case in a serial biopsy after 7 years of transplant survival. The other serial biopsies, as well as the final sections at autopsy, showed no recipient pattern chromosomes within the myocytes. In addition, 2 cases demonstrated Y chromosomes in the smooth muscle of intracardiac arteries. Conclusion Development of recipient marrow–derived stem cells into functional myocytes in the ventricle of transplanted hearts is a rare feature. The higher percentage of stem cell population in the cardiac atria may be a feature of tissue repair and/or an early feature of transplant physiology. Similar to other transplant organs, recipient stem cells are involved in tissue neovascularization.

Efficient gene delivery and gene expression in zebrafish using the Sleeping Beauty transposon

We used the Tc1/mariner family transposable element Sleeping Beauty (SB) for transgenesis and long-term expression studies in the zebrafish ( Danio rerio ), a popular organism for clinical disease, vertebrate patterning, and cell biology applications. SB transposase enhanced the transgenesis and expression rate sixfold (from 5 to 31%) and more than doubled the total number of tagged chromosomes over standard, plasmid injection-based transgenesis methods. Molecular analysis of these loci demonstrated a precise integration of these elements into recipient chromosomes with genetic footprints diagnostic of transposition. GFP expression from transposase-mediated integrants was Mendelian through the eighth generation. A blue-shifted GFP variant (BFP) and a red fluorescent protein (DsRed) were also useful transgenesis markers, indicating that multiple reporters are practical for use with SB in zebrafish. We showed that SB is suitable for tissue-specific transgene applications using an abbreviated gamma-crystallin GFP cassette. Finally, we describe a general utility transposon vector for chromosomal engineering and molecular genetics experiments in zebrafish. Together, these data indicate that SB is an efficient tool for transgenesis and expression in zebrafish, and that the transposon will be useful for gene expression in cell biology applications as well as an insertional mutagen for gene discovery during development.

Efficient gene delivery and gene expression in zebrafish using the Sleeping Beauty transposon

We used the Tc1/mariner family transposable element Sleeping Beauty (SB) for transgenesis and long-term expression studies in the zebrafish ( Danio rerio), a popular organism for clinical disease, vertebrate patterning, and cell biology applications. SB transposase enhanced the transgenesis and expression rate sixfold (from 5 to 31%) and more than doubled the total number of tagged chromosomes over standard, plasmid injection-based transgenesis methods. Molecular analysis of these loci demonstrated a precise integration of these elements into recipient chromosomes with genetic footprints diagnostic of transposition. GFP expression from transposase-mediated integrants was Mendelian through the eighth generation. A blue-shifted GFP variant (BFP) and a red fluorescent protein (DsRed) were also useful transgenesis markers, indicating that multiple reporters are practical for use with SB in zebrafish. We showed that SB is suitable for tissue-specific transgene applications using an abbreviated gamma-crystallin GFP cassette. Finally, we describe a general utility transposon vector for chromosomal engineering and molecular genetics experiments in zebrafish. Together, these data indicate that SB is an efficient tool for transgenesis and expression in zebrafish, and that the transposon will be useful for gene expression in cell biology applications as well as an insertional mutagen for gene discovery during development.

Unconventional conjugal DNA transfer in mycobacteria.

Bacterial conjugation is an active process that results in unidirectional transfer of DNA from a donor to a recipient cell. Most transfer systems are plasmid-encoded and require proteins to act at a unique cis-acting site to initiate and complete DNA transfer. By contrast, the Mycobacterium smegmatis DNA transfer system is chromosomally encoded. Here we show that multiple cis-acting sequences present on the chromosome can mediate transfer of a non-mobilizable test plasmid. Moreover, unlike conventional plasmid transfer, recipient recombination functions are required to allow this plasmid, and derivatives of it, to re-circularize through a process similar to gap repair. Extended DNA homology with the recipient chromosome is required to facilitate repair, resulting in acquisition of recipient chromosomal DNA by the plasmid. Together, these results show that DNA transfer in M. smegmatis occurs by a mechanism different from that of prototypical plasmid transfer systems.

Combined 24-color karyotyping and comparative genomic hybridization analysis indicates predominant rearrangements of early replicating chromosome regions in neuroblastoma

Neuroblastoma is characterized by several distinct genetic alterations including MYCN amplification, chromosome 1p deletion and gain of chromosome 17. Although these alterations are thought to play a crucial role in oncogenesis, to date little is known about their underlying mechanisms. In order to more precisely document these genetic alterations, we have performed a combined study of 27 neuroblastoma cell lines using 24-color karyotyping (24-CK) and comparative genomic hybridization (CGH). 24-CK detected balanced translocations in 13 cases with recurrent involvement of chromosome 8. More importantly, 144 nonreciprocal translocations were observed in the 27 cell lines, with chromosome 1 as the most frequent recipient and chromosome 17 the most frequent donor. Each cell line exhibited at least one unbalanced translocation involving 17q, with 14 cell lines demonstrating more than one such translocation. Other recurrent alterations were amplification of the 2p24 chromosome region, which encodes the MYCN oncogene, losses of 1p, 3p and 11q, and gains of 1q and 7. In most cases, CGH profiles were directly linked to the presence of unbalanced translocations with gain of the donor fragment and loss of the replaced region on the recipient chromosome. Strikingly, over 60% of the chromosome breakpoints mapped to early replicating chromosome bands, which represent around 13% of the genome. Altogether these data suggest that neuroblastoma is characterized by rearrangements that predominantly involve chromosome fragments replicating early in the S-phase.

Intergeneric conjugation in Streptomyces peucetius and Streptomyces sp. strain C5: chromosomal integration and expression of recombinant plasmids carrying the chiC gene.

Intergeneric conjugal transfer of plasmid DNA from Escherichia coli to Streptomyces circumvents problems such as host-controlled restriction and instability of foreign DNA during the transformation of Streptomyces protoplasts. The anthracycline antibiotic-producing strains Streptomyces peucetius and Streptomyces sp. strain C5 were transformed using E. coli ET12567(pUZ8002) as a conjugal donor. When this donor species, carrying pSET152, was mated with Streptomyces strains, the resident plasmid was mobilized to the recipient and the transferred DNA was also integrated into the recipient chromosome. Analysis of the exconjugants showed stable integration of the plasmid at a single chromosomal site (attB) of the Streptomyces genome. The DNA sequence of the chromosomal integration site was determined and shown to be conserved. However, the core sequence, where the crossover presumably occurred in C5 and S. peucetius, is TTC. These results also showed that the phiC31 integrative recombination is active and the phage attP site is functional in S. peucetius as well as in C5. The efficiency and specificity of phiC31-mediated site-specific integration of the plasmid in the presence of a 3.7-kb homologous DNA sequence indicates that integrative recombination is preferred under these conditions. The integration of plasmid DNA did not affect antibiotic biosynthesis or biosynthesis of essential amino acids. Integration of a single copy of a mutant chiC into the wild-type S. peucetius chromosome led to the production of 30-fold more chitinase.

Structure-Function Analysis of Escherichia coli DNA Helicase I Reveals Non-overlapping Transesterase and Helicase Domains

TraI (DNA helicase I) is an Escherichia coli F plasmid-encoded protein required for bacterial conjugative DNA transfer. The protein is a sequence-specific DNA transesterase that provides the site- and strand-specific nick required to initiate DNA strand transfer and a 5' to 3' DNA helicase that unwinds the F plasmid to provide the single-stranded DNA that is transferred from donor to recipient. Sequence comparisons with other transesterases and helicases suggest that these activities reside in the N- and C-terminal regions of TraI, respectively. Computer-assisted secondary structure probability analysis identified a potential interdomain region spanning residues 304-309. Proteins encoded by segments of traI, whose N or C terminus either flanked or coincided with this region, were purified and assessed for catalytic activity. Amino acids 1-306 contain the transesterase activity, whereas amino acids 309-1504 contain the helicase activity. The C-terminal 252 amino acids of the 1756-amino acid TraI protein are not required for either helicase or transesterase activity. Protein and nucleic acid sequence similarity searches indicate that the occurrence of both transesterase- and helicase-associated motifs in a conjugative DNA transfer initiator protein is rare. Only two examples (other than R100 plasmid TraI) were found: R388 plasmid TrwC and R46 plasmid (pKM101) TraH, belonging to the IncW and IncN groups of broad host range conjugative plasmids, respectively. The most significant structural difference between these proteins and TraI is that TraI contains an additional region of approximately 650 residues between the transesterase domain and the helicase-associated motifs. This region is required for helicase activity.

Application of mFISH for the analysis of chemically-induced chromosomal aberrations: a model for the formation of triradial chromosomes

Using a human lymphoblastoid cell line WTK-1, we applied multicolor fluorescence in situ hybridization (mFISH) technique to analyze mitomycin C (MMC)-induced chromatid exchanges, focusing especially on the triradial chromosomes. It was found that the triradial chromosomes were formed with a specific rearrangement, “recipient and donor” relationship. The exchange sites of the recipient chromosomes were on single chromatid breaks and distributed randomly throughout the interstitial, pericentromeric, and terminal regions. In counterpart, donor chromosomes exchanged on isochromatid breaks of their telomeric and/or subtelomeric regions with the single chromatid breaks of recipient chromosomes. More than 80% of the scored triradial chromosomes were formed with such rearrangements, and few acentric chromosome fragments derived from the donor chromosomes could be detected in the metaphases observed. We therefore suggest that biological mechanisms of breakages between the recipient and donor chromosomes are different: the former due to direct DNA-damage by MMC, but the latter due to indirect DNA-damage depending on telomeric specific structure/function.

Attempts to Transfer Russian Wheat Aphid Resistance from a Rye Chromosome in Russian Triticales to Wheat

The Russian wheat aphid (Diuraphis noxia Mordvilko) is a serious pest of wheat (Triticum aestivum L.). To extend the range of genetic variation of resistance, attempts were undertaken to transfer near‐immunity to RWA into wheat from two Russian triticales (X. Triticosecale Wittmack) PI 386146 and PI 386156 by irradiation and by induced homologous recombination. The rye genome in the triticale lines was derived from Secale montanum Guss. Tests of resistance in early backcrosses to wheat indicated that the near‐immunity of the triticale lines was controlled by at least two loci, one of which was located on rye chromosome arm 4RLmon Centric wheat‐rye translocation 7DS.4RLmon that appeared to be compensating, was produced. To further reduce the amount of rye chromatin present, its long arm was induced to recombine with wheat chromosomes by the removal of the Ph1 locus. Among 3563 progeny screened, only two wheat‐rye recombinant chromosomes were recovered. Both appeared to be non‐compensating and were involved in multivalents in meiosis. Irradiation of PI 386156 followed by crosses and backcrosses to wheat with several generations of selection for resistance resulted in a wheat line that was found to be a disomic addition of chromosome 4Rmon–centric translocation homozygote of rye chromosome tentatively identified as 5Rmon With only one locus for resistance from the original triticale parents, the addition line of 4Rmon, centric translocation line 7DS.4RLmon and recombinant lines of 4RLmon had only moderate level of resistance to RWA. The study demonstrates that transfers of alien variation into wheat may be severely complicated by unclear genetics of the target traits, low levels of homology, and structural differences between the donor and recipient chromosomes.

Extramedullary T lymphoid blast crisis representing an additional translocation, t(6;8)(q25;q22) in a patient with Philadelphia-positive chronic myelogenous leukemia after allogeneic bone marrow transplantation

A patient with extramedullary crisis from chronic myelogenous leukemia after allogeneic bone marrow transplantation is reported. A pathological neck lymph node observed after transplantation revealed pre-T lymphoblastic phenotype, and the fluorescence in situ hybridization (FISH) analysis showed recipient type sex chromosomes and bcr/abl fusion gene. The cells represented an additional translocation, t(6;8)(q25;q22). No rearrangements of the T-cell receptor (TCR) β, γ or δ chain genes were observed. The absence of TCR rearrangement indicated the clonogenic involvement of pluripotent hematopoietic stem cells by Philadelphia chromosome. Bone marrow specimens at that time showed donor type sex chromosomes and no bcr/abl-positive cells by FISH.

A method to maintain introduced DNA sequences stably and safely on the bacterial chromosome: Application of prophage integration and subsequent designed excision

By application of prophage integration and subsequent intended excision, a method to maintain an introduced DNA sequence stably onto a bacterial chromosome has been proposed. Recently-constructed integration plasmids using Campbell-type prophage integration system in Lactobacillus casei strain Shirota and its temperate phage φFSW was modified for this purpose and a chloramphenicol (Cm)-resistance gene was used as a model passenger DNA. On the integration plasmid having an erythromycin (Em)-resistance gene as a selection marker, N- and C-terminally-truncated Cm-resistance genes were inserted into both sides of the attP of φFSW, within which the site-specific recombination took place with the attB of φFSW on the recipient chromosome through the φFSW integrase. Primary integrants of the modified plasmid (integration-excision vector) exhibiting Em-resistant and Cm-sensitive phenotype generated Em-sensitive and Cm-resistant derivatives under the nonselective conditions. Sequence analyses showed that one copy of the complete Cm-resistance gene resided at the attachment site on the host chromosome and the other vector-derived sequences were excised probably by endogenous homologous recombination in the host cells to derive final integrants. The Cm-resistant phenotype of the final integrants was stable for more than 50 generations under non-selective conditions. Frequency of the homologous recombination suggests that negative selection is also adoptable. Thus, this method using the integration-excision vector gives a stable and safe derivatives of the strain and is likely to be applicable to various bacteria, since Campbell-type prophage integration system and homologous recombination are prevalent among bacteria.

Transformation of an oral bacterium via chromosomal integration of free DNA in the presence of human saliva

Transformation of Streptococcus gordonii DL1 by free DNA was studied in human saliva. Competent S. gordonii could be transformed in vitro with plasmid DNA that had been taken into the human mouth. Transformation also occurred with a plasmid that cannot replicate in S. gordonii, but that has a region of chromosomal homology, by integration into the bacterial chromosome, although linearised plasmid DNA gave no transformants. Linear chromosomal DNA fragments did however transform S. gordonii/Tn 916 efficiently in saliva when regions of homology with the recipient chromosome flanked the marker gene. These findings are discussed in relation to the potential for acquisition of DNA sequences, including genetically modified DNA, by gut and oral bacteria.

Transformation of an oral bacterium via chromosomal integration of free DNA in the presence of human saliva.

Transformation of Streptococcus gordonii DL1 by free DNA was studied in human saliva. Competent S. gordonii could be transformed in vitro with plasmid DNA that had been taken into the human mouth. Transformation also occurred with a plasmid that cannot replicate in S. gordonii, but that has a region of chromosomal homology, by integration into the bacterial chromosome, although linearised plasmid DNA gave no transformants. Linear chromosomal DNA fragments did however transform S. gordonii/Tn916 efficiently in saliva when regions of homology with the recipient chromosome flanked the marker gene. These findings are discussed in relation to the potential for acquisition of DNA sequences, including genetically modified DNA, by gut and oral bacteria.

Jumping translocations are common in solid tumor cell lines and result in recurrent fusions of whole chromosome arms.

Jumping translocations (JTs) and segmental jumping translocations (SJTs) are unbalanced translocations involving a donor chromosome arm or chromosome segment that has fused to multiple recipient chromosomes. In leukemia, where JTs have been predominantly observed, the donor segment (usually 1q) preferentially fuses to the telomere regions of recipient chromosomes. In this study, spectral karyotyping (SKY) and FISH analysis revealed 188 JTs and SJTs in 10 cell lines derived from carcinomas of the bladder, prostate, breast, cervix, and pancreas. Multiple JTs and SJTs were detected in each cell line and contributed to recurrent unbalanced whole-arm translocations involving chromosome arms 5p, 14q, 15q, 20q, and 21q. Sixty percent (113/188) of JT breakpoints occurred within centromere or pericentromeric regions of the recipient chromosomes, whereas only 12% of the breakpoints were located in the telomere regions. JT breakpoints of both donor and recipient chromosomes coincided with numerous fragile sites as well as viral integration sites for human DNA viruses. The JTs within each tumor cell line promoted clonal progression, leading to the acquisition of extra copies of the donated chromosome segments that often contained oncogenes (MYC, ABL, HER2/NEU, etc.), consequently resulting in tumor-specific genomic imbalances. Published 2001 Wiley-Liss, Inc.