Human Chromosome Fragments Research Articles

Rapid human genomic DNA cloning into mouse artificial chromosome via direct chromosome transfer from human iPSC and CRISPR/Cas9-mediated translocation.

A 'genomically' humanized animal stably maintains and functionally expresses the genes on human chromosome fragment (hCF; <24 Mb) loaded onto mouse artificial chromosome (MAC); however, cloning of hCF onto the MAC (hCF-MAC) requires a complex process that involves multiple steps of chromosome engineering through various cells via chromosome transfer and Cre-loxP chromosome translocation. Here, we aimed to develop a strategy to rapidly construct the hCF-MAC by employing three alternative techniques: (i) application of human induced pluripotent stem cells (hiPSCs) as chromosome donors for microcell-mediated chromosome transfer (MMCT), (ii) combination of paclitaxel (PTX) and reversine (Rev) as micronucleation inducers and (iii) CRISPR/Cas9 genome editing for site-specific translocations. We achieved a direct transfer of human chromosome 6 or 21 as a model from hiPSCs as alternative human chromosome donors into CHO cells containing MAC. MMCT was performed with less toxicity through induction of micronucleation by PTX and Rev. Furthermore, chromosome translocation was induced by simultaneous cleavage between human chromosome and MAC by using CRISPR/Cas9, resulting in the generation of hCF-MAC containing CHO clones without Cre-loxP recombination and drug selection. Our strategy facilitates rapid chromosome cloning and also contributes to the functional genomic analyses of human chromosomes.

An optimized proliferation system of embryonic stem cells for generating the rat model with large fragment modification

Rats have long been an ideal model for disease research in the field of biomedicine, but the bottleneck of in vitro culture of rat embryonic stem (ES) cells hindered the wide application as genetic disease models. Here, we optimized a special medium which we named 5N-medium for rat embryonic stem cells, which improved the in vitro cells with better morphology and higher pluripotency. We then established a drug selection schedule harboring a prior selection of 12 h that achieved a higher positive selection ratio. These treatments induced at least 50% increase of homologous recombination efficiency compared with conventional 2i culture condition. Moreover, the ratio of euploid ES clones also increased by 50% with a higher germline transmission rate. Finally, we successfully knocked in a 175 kb human Bacterial Artificial Chromosome (BAC) fragment to rat ES genome through recombinase mediated cassette exchange (RMCE). Hence, we provide a promising system for generating sophisticated rat models which could be benefit for biomedical researches.

Reactivation of heat-inactivated Ku proteins by heat shock cognate protein HSC73

Purpose: Mouse double-stranded DNA-dependent protein kinase (DNA-PK) activity is heat sensitive. Recovery of heat-inactivated DNA repair activity is a problem after combination therapy with radiation and heat. We investigated the mechanism of recovery of heat-inactivated DNA-PK activity.Methods: Hybrid cells containing a fragment of human chromosome 8 in scid cells (RD13B2) were used. DNA-PK activity was measured by an in vitro assay. Immunoprecipitation of the nuclear extract was performed with an anti-Ku80 antibody. Proteins co-precipitated with Ku80 were separated by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and detected by Western blotting using anti-heat shock protein (HSP)72 and anti-heat shock cognate protein (HSC)73 antibodies. HSC73 was overexpressed with the pcDNA3.1 vector. Short hairpin (sh)RNA was used to downregulate HSC73 and HSP72.Results: The activity of heat-inactivated DNA-PK recovered to about 50% of control during an additional incubation at 37 °C after heat treatment at 44 °C for 15 min in the presence of cycloheximide (which inhibits de novo protein synthesis). Maximal recovery was observed within 3 h of incubation at 37 °C after heat treatment. Constitutively expressed HSC73, which folds newly synthesized proteins, reached maximal levels 3 h after heat treatment using a co-immunoprecipitation assay with the Ku80 protein. Inhibiting HSC73, but not HSP72, expression with shRNA decreased the recovery of DNA-PK activity after heat treatment.Conclusions: These results suggest that de novo protein synthesis is unnecessary for recovery of some heat-inactivated DNA-PK. Rather, it might be reactivated by the molecular chaperone activity of HSC73, but not HSP72.

New possibilities of effective breeding in cattle based on the study of the genome

The article contains generalized literary data on the results of genome research based on molecular genetic methods in connection with the productive qualities of cattle that can be used to accelerate and improve breeding work. The study of the human genome has given impetus to the development of medicine, biotechnology and pharmacogenetics. Similarly, new research on the genome of cattle gives qualitatively different possibilities for using these data in the selection and production of agricultural products, as well as in controlling its quality. Molecular genetic markers inform about the polymorphism of genes and allow to detect individual genes and gene complexes that carry information about a certain feature. Based on such studies, gene pool can be formed with a certain combination. An alternative way of molecular-genetic marking of performance is to study the polymorphism of structural genes, allelic variants which are directly related to the desired phenotypic manifestation, namely: kappa-casein (CSN3), veta-lactaglobulin (BLG), somatotropin (GH), and myostatin (MSTN). Modern breeding work with cattle is associated with the establishment of a connection between the polygenic signs of productivity and the «main» genes of quantitative traits, the polymorphism of which affects the final output of the protein product. As candidate genes that affect lactation productivity in cattle, first of all the genes of milk proteins, in particular kappa-casein, are examined. The gene for the somatotropic hormone (GH), a growth hormone in cattle, is a polypeptide consisting of 191 amino acids and is encoded by a single gene, which is localized in 19 chromosomes. Growth hormone plays a key role in stimulating the synthesis of protein, cell division, and body growth. Myostatin – one of the regulators of skeletal muscle development is the myostatin gene, which refers to a family of transforming growth factors. The gene of myostatin in the Bovine species is localized in chromosome 2 and carries the muscle hypertrophy locus, there is also a homologous fragment of human chromosome 2, where the locus of this gene is limited. The presence of the gene of myostatin, as one of the locus of quantitative traits of beef, can be used as a marker for genetic mapping. After discovering mutations in the gene of the myostatin, they came to the conclusion, that it is not the only gene that controls the growth and muscle mass of animals. Molecular genetic markers allow you to receive information about the polymorphism of genes and to identify individual genes and gene «ensembles» that carry the desired complex of features.

Human monoclonal antibody GNX-8 directed to extended type 1 chain: Specific binding to human colorectal cancer.

We observed previously that two carbohydrate epitopes, extended type 1 chain Le(a)-Le(a) and Le(b)-Le(a), are expressed strongly in human gastric or colorectal cancer and cell lines derived therefrom, but their expression in human normal colorectal cells is highly limited. A monoclonal antibody, termed GNX-8, was established through immunization of "KM mice" with colonic cancer cell line Colo205, and with purified Le(b)-Le(a) glycosphingolipid, followed by screening human IgG directed to this antigen. KM mice possess human chromosome fragments and are capable of producing human immunoglobulin. GNX-8 reacted specifically with extended type 1 chain epitope Le(b)-Le(a), bound to all five colonic cancer cell lines so far tested, and displayed strong complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC). The antigens defined by GNX-8, expressed in Colo205 cells, were: (i) glycosphingolipids with epitope Le(b)-Le(a), whose reactivity was abolished upon defucosylation; (ii) glycoproteins with molecular mass range from 32 to >175 kDa, which were depleted in cells cultured in the presence of benzyl-alpha-GalNAc, indicating that these epitopes are O-linked glycans.Immunohistological reactivity of GNX-8 at 1 mug/ml, applied on tissue sections from colorectal and various other types of cancer, was much stronger than that with various normal cells and tissues. GNX-8 reactivity with normal cells required a much higher concentration (150 mug/ml), and this reactivity was based on cross-reaction with non-extended, normal blood group Le(b) antigen. Growth of subcutaneous xenograft of human colonic cancer cells, Colo205 or DLD-1, in nude mice or SCID mice, was strongly inhibited by administration of GNX-8. These observations, taken together, indicate that antibody GNX-8, directed specifically to Le(b)-Le(a) antigen, provides a novel direction of immunotherapy for human colorectal cancer. (c) 2009 UICC.

Human antibody production in transgenic animals.

Fully human antibodies from transgenic animals account for an increasing number of new therapeutics. After immunization, diverse human monoclonal antibodies of high affinity can be obtained from transgenic rodents, while large animals, such as transchromosomic cattle, have produced respectable amounts of specific human immunoglobulin (Ig) in serum. Several strategies to derive animals expressing human antibody repertoires have been successful. In rodents, gene loci on bacterial artificial chromosomes or yeast artificial chromosomes were integrated by oocyte microinjection or transfection of embryonic stem (ES) cells, while ruminants were derived from manipulated fibroblasts with integrated human chromosome fragments or human artificial chromosomes. In all strains, the endogenous Ig loci have been silenced by gene targeting, either in ES or fibroblast cells, or by zinc finger technology via DNA microinjection; this was essential for optimal production. However, comparisons showed that fully human antibodies were not as efficiently produced as wild-type Ig. This suboptimal performance, with respect to immune response and antibody yield, was attributed to imperfect interaction of the human constant region with endogenous signaling components such as the Igα/β in mouse, rat or cattle. Significant improvements were obtained when the human V-region genes were linked to the endogenous CH-region, either on large constructs or, separately, by site-specific integration, which could also silence the endogenous Ig locus by gene replacement or inversion. In animals with knocked-out endogenous Ig loci and integrated large IgH loci, containing many human Vs, all D and all J segments linked to endogenous C genes, highly diverse human antibody production similar to normal animals was obtained.

A novel transchromosomic system: stable maintenance of an engineered Mb-sized human genomic fragment translocated to a mouse chromosome terminal region

Transchromosomic (Tc) technology using human chromosome fragments (hCFs), or human artificial chromosomes (HACs), has been used for generating mice containing Mb-sized segments of the human genome. The most significant problem with freely segregating chromosomes with human centromeres has been mosaicism, possibly due to the instability of hCFs or HACs in mice. We report a system for the stable maintenance of Mb-sized human chromosomal fragments following translocation to mouse chromosome 10 (mChr.10). The approach utilizes microcell-mediated chromosome transfer and a combination of site-specific loxP insertion, telomere-directed chromosome truncation, and precise reciprocal translocation for the generation of Tc mice. Human chromosome 21 (hChr.21) was modified with a loxP site and truncated in homologous recombination-proficient chicken DT40 cells. Following transfer to mouse embryonic stem cells harboring a loxP site at the distal region of mChr.10, a ~4Mb segment of hChr.21 was translocated to the distal region of mChr.10 by transient expression of Cre recombinase. The residual hChr.21/mChr.10ter fragment was reduced by antibiotic negative selection. Tc mice harboring the translocated ~4Mb fragment were generated by chimera formation and germ line transmission. The hChr.21-derived Mb fragment was maintained stably in tissues in vivo and expression profiles of genes on hChr.21 were consistent with those seen in humans. Thus, Tc technology that enables translocation of human chromosomal regions onto host mouse chromosomes will be useful for studying in vivo functions of the human genome, and generating humanized model mice.

A Novel and Stable Mouse Artificial Chromosome Vector

Human chromosome fragments (hCFs) and human artificial chromosomes (HACs) can be transferred into mouse ES cells to produce trans-chromosomic (Tc) mice. Although hCFs and HACs containing large genomic DNAs can be autonomously maintained in Tc mice, their retention rate is variable in mouse ES cell lines and Tc mouse tissues, possibly because of centromere differences between the species. To improve the retention rate of artificial chromosomes in mouse cells, we constructed novel mouse artificial chromosome (MAC) vectors by truncating a natural mouse chromosome at a site adjacent to the centromeric region. We obtained cell clones containing the MAC vectors that were stably maintained in mouse ES cells and various tissues in Tc mice. The MACs possess acceptor sites into which a desired gene or genes can be inserted. Thus, Tc mice harboring the MAC vectors may be valuable tools for functional analyses of desired genes, producing humanized model mice, and synthetic biology.

Transfer of human artificial chromosome vectors into stem cells

Human chromosome fragments and human artificial chromosomes (HAC) represent feasible gene delivery vectors via microcell-mediated chromosome transfer. Strategies to construct HAC involve either ‘build up’ or ‘top-down’ approaches. For each approach, techniques for manipulating HAC in donor cells in order to deliver HAC to recipient cells are required. The combination of chromosome fragments or HAC with microcell-mediated chromosome transfer has facilitated human gene mapping and various genetic studies. The recent emergence of stem cell-based tissue engineering has opened up new avenues for gene and cell therapies. The task now is to develop safe and effective vectors that can deliver therapeutic genes into specific stem cells and maintain long-term regulated expression of these genes. Although the transfer-efficiency needs to be improved, HAC possess several characteristics that are required for gene therapy vectors, including stable episomal maintenance and the capacity for large gene insets. HAC can also carry genomic loci with regulatory elements, which allow for the expression of transgenes in a genetic environment similar to the natural chromosome. This review describes the lessons and prospects learned, mainly from recent studies in developing HAC and HAC-mediated gene expression in embryonic and adult stem cells, and in transgenic animals.

Isolation and characterization of mouse–human microcell hybrid cell clones permissive for infectious HIV particle release

Mouse cells are non-permissive to human immunodeficiency virus type 1 (HIV) in that there is a pronounced post-integration block to viral replication. We have recently demonstrated that mouse–human somatic cell hybrids that contain human chromosome 2 increase both HIV Capsid (CA) production and infectious virus release. Here we report on the isolation of three mouse–human microcell hybrids (MCHs) that behave similarly, starting from a pool of 500 MCH clones. Release of virus was specific to HIV and cell revertants that no longer contained any human chromosome fragments did not release CA or infectious virus. Two of the three cell clones were identical as judged by PCR STS content and fluorescence in situ hybridization (FISH) and contained a single 2–12 human chromosome chimera. The third cell clone only contained human chromosome 12, as determined by PCR, FISH, and microarray analyses. There were no consistent differences in Gag protein and spliced/unspliced viral RNA levels between mouse cell lines. CMV promoter-driven, codon-optimized gag-pol had no effect on infectious HIV release from these mouse cells, despite allowing Gag targeting and increasing CA production. These permissive mouse–human MCHs and their corresponding non-permissive revertants may prove useful for mechanistic studies and also for identifying the responsible gene(s) or factor(s) involved in the production of HIV.

Identification of a Novel Tumor Suppressor Gene p34 on Human Chromosome 6q25.1

In this study, we observed loss of heterozygosity (LOH) in human chromosomal fragment 6q25.1 in sporadic lung cancer patients. LOH was observed in 65% of the 26 lung tumors examined and was narrowed down to a 2.2-Mb region. Single-nucleotide polymorphism (SNP) analysis of genes located within this region identified a candidate gene, termed p34. This gene, also designated as ZC3H12D, C6orf95, FLJ46041, or dJ281H8.1, carries an A/G nonsynonymous SNP at codon 106, which alters the amino acid from lysine to arginine. Nearly 73% of heterozygous lung cancer tissues with LOH and the A/G SNP also exhibited loss of the A allele. In vitro clonogenic and in vivo nude mouse studies showed that overexpression of the A allele exerts tumor suppressor function compared with the G allele. p34 is located within a recently mapped human lung cancer susceptibility locus, and association of the p34 A/G SNP was tested among these families. No significant association between the less frequent G allele and lung cancer susceptibility was found. Our results suggest that p34 may be a novel tumor suppressor gene involved in sporadic lung cancer but it seems not to be the candidate familial lung cancer susceptibility gene linked to chromosomal region 6q23-25.

High resolution physical map of porcine chromosome 7 QTL region and comparative mapping of this region among vertebrate genomes

BackgroundOn porcine chromosome 7, the region surrounding the Major Histocompatibility Complex (MHC) contains several Quantitative Trait Loci (QTL) influencing many traits including growth, back fat thickness and carcass composition. Previous studies highlighted that a fragment of ~3.7 Mb is located within the Swine Leucocyte Antigen (SLA) complex. Internal rearrangements of this fragment were suggested, and partial contigs had been built, but further characterization of this region and identification of all human chromosomal fragments orthologous to this porcine fragment had to be carried out.ResultsA whole physical map of the region was constructed by integrating Radiation Hybrid (RH) mapping, BAC fingerprinting data of the INRA BAC library and anchoring BAC end sequences on the human genome. 17 genes and 2 reference microsatellites were ordered on the high resolution IMNpRH212000rad Radiation Hybrid panel. A 1000:1 framework map covering 550 cR12000 was established and a complete contig of the region was developed. New micro rearrangements were highlighted between the porcine and human genomes. A bovine RH map was also developed in this region by mapping 16 genes. Comparison of the organization of this region in pig, cattle, human, mouse, dog and chicken genomes revealed that 1) the translocation of the fragment described previously is observed only on the bovine and porcine genomes and 2) the new internal micro rearrangements are specific of the porcine genome.ConclusionWe estimate that the region contains several rearrangements and covers 5.2 Mb of the porcine genome. The study of this complete BAC contig showed that human chromosomal fragments homologs of this heavily rearranged QTL region are all located in the region of HSA6 that surrounds the centromere. This work allows us to define a list of all candidate genes that could explain these QTL effects.

The Trans‐Chromosomic Mouse‐Derived Human Monoclonal Antibody Promotes Phagocytosis of Porphyromonas gingivalis by Neutrophils

As a safe immunotherapeutic approach, human monoclonal antibody (hMAb) may be effective in clearing periodontopathic bacteria. The trans-chromosomic (TC) technology has recently been applied to construction of the TC mouse, which enables us to incorporate entire human chromosome fragments containing immunoglobulin (Ig) gene cluster. The aim of this study is to establish TC mouse-derived hMAb, and to test the in vitro opsonophagocytic activity. Human Ig-producing TC mouse was immunized by recombinant 40-kDa outer membrane protein (r40-kDa OMP) of Porphyromonas gingivalis 381, and the spleen cells were fused with the mouse myeloma cell line. The specificity of antir40- kDa OMP hMAb was evaluated with the enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance assays. Flow cytometric analyses were performed to assess the opsonophagocytic activity. We successfully constructed 99 IgG isotype clones (IgG1: 84; IgG2: 11; IgG4: four clones), which were specifically reactive with r40-kDa OMP. The anti-r40-kDa OMP IgG1 hMAbs promoted phagocytosis of P. gingivalis by neutrophils. Futhermore, an increased opsonophagocytic activitity of anti-r40-kDa OMP IgG1 hMAbs was observed not only in P. gingivalis 381, but also in the W50, W83, and Su63 strains. Our results document the TC mouse-derived hMAb to promote neutrophil phagocytosis of P. gingivalis, suggesting an immunotherapeutic option for clearance of P. gingivalis.

A temperature-sensitive mutation in the WD repeat-containing protein Smu1 is related to maintenance of chromosome integrity

Temperature-sensitive CHO-K1 mutant cell line tsTM18 exhibits chromosomal instability and cell cycle arrest at S and G 2 phases with decreased DNA synthesis at the nonpermissive temperature, 39°C. To identify the causative mutation, we fused tsTM18 cells with normal human cells to generate hybrids carrying fragments of human chromosomes. Analysis of chromosome content of temperature-resistant transformants and introduction of a bacterial artificial chromosome containing part of human chromosome 9 led to isolation of the human SMU1 gene. Comparison of sequences of the Smu1 gene from wild-type and mutant cells revealed that the mutant phenotype is caused by a G-to-A transition that yields a gly-to-arg substitution at position 489 in hamster Smu1. The substituted glycine is located in the WD-repeat domain of Smu1. Single-stranded DNA accumulated in the nuclei of mutant cells at 39°C. Furthermore, cdc2 kinase was not activated during G 2 phase, and there was no chromosome segregation due to incomplete assembly of the spindle during M phase. Thus, Smu1 appears to be involved directly or indirectly in DNA replication, activation of cdc2 kinase, spindle assembly, and maintenance of chromosome integrity, reflecting the important roles of Smu1 in cellular function.

Analysis of Mitotic and Expression Properties of Human Neocentromere-based Transchromosomes in Mice

Human neocentromeres are functional centromeres that are devoid of the typical human centromeric alpha-satellite DNA. We have transferred a 60-Mb chromosome 10-derived neocentric marker chromosome, mardel(10), and its truncated 3.5-Mb derivative, NC-MiC1, into mouse embryonic stem cell and have demonstrated a relatively high structural and mitotic stability of the transchromosomes in a heterologous genetic background. We have also produced chimeric mice carrying mardel(10) or NC-MiC1. Both transchromosomes were detected as intact episomal entities in a variety of adult chimeric mouse tissues including hemopoietic stem cells. Genes residing on these transchromosomes were expressed in the different tissues tested. Meiotic transmission of both transchromosomes in the chimeric mice was evident from the detection of DNA from these chromosomes in sperm samples. In particular, germ line transmission of NC-MiC1 was demonstrated in the F1 embryos of the chimeric mice. Variable (low in mardel(10)- or NC-MiC1-containing embryonic stem cells and chimeric mouse tissues and relatively high in NC-MiC1-containing F1 embryos) levels of missegregation of these transchromosomes were detected, suggesting that they are not optimally predisposed to full mitotic regulation in the mouse background, particularly during early embryogenesis. These results provide promising data in support of the potential use of neocentromere-based human marker chromosomes and minichromosomes as a tool for the study of centromere, neocentromere, and chromosome biology and for gene therapy studies in a mouse model system. They also highlight the need to further understand and overcome the factors that are responsible for the definable rates of instability of these transchromosomes in a mouse model.

Human Monoclonal Antibodies to TRAIL Receptors R1 and R2 Effectively Induce Apoptosis in Human Myeloma Cells.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent activator of apoptotic pathway in a variety of tumor cells but not normal cells. Therefore, TRAIL and its receptors have been considered as possible therapeutic targets in cancer treatment. However, several myeloma cells were resistant to TRAIL-induced apoptosis depending on the expression patterns of TRAIL receptors including TRAIL-R1 and -R2 death receptors, and TRAIL-R3 and -R4 decoy receptors. To explore the contribution of each TRAIL receptor to apoptosis induction of myeloma cells, we generated fully human monoclonal antibodies (MoAbs) that bind specifically to TRAIL-R1 and TRAIL-R2 using KM mice that possess the human chromosome fragments containing human immunoglobulin heavy chain loci and YAC transgene containing human kappa light chain gene. Several myeloma cell lines as well as freshly isolated myeloma cells were cultured with TRAIL or these MoAbs in the presence of F(ab′)2 goat anti-human IgG. Soluble TRAIL (1000 ng/mL) showed more than 80% of cytotoxicity in RPMI 8226 and KMS12-BM cells within 24 hours. However, U266, HS-Sultan, and ARH-77 cells were relatively resistant to TRAIL with maximal cytotoxicity of only 3–31%. In contrast, anti-TRAIL-R1 MoAb (R1-B12) effectively induced apoptosis even in TRAIL-resistant myeloma cell lines in a time- and dose-dependent manner with maximal cytotoxicity of 65-99%. This apoptotic response of myeloma cells was confirmed by caspase activation and annexin V binding. On the other hand, anti-TRAIL-R2 MoAb (R2-E11) was less effective against these myeloma cell lines showing 1–33% of cytotoxicity. Among 10 primary myeloma cells, R1-B12 induced at least 10% of cytotoxicity in 7 samples and R2-E11 showed in 3 samples. Flow cytometric analysis demonstrated that TRAIL-R1 was expressed at a higher level than TRAIL-R2 on these myeloma cell lines and specific mean fluorescence intensity (MFI) was 3.8–7.9 and 1.6–4.2, respectively. TRAIL-R1 and -R2 were also detected on primary myeloma cells at low levels and specific MFI was 1.0–2.0 and 1.0–1.6, respectively. Thus, the sensitivity to R1-B12 and R2-E11 was correlated with the expression level of TRAIL-R1 and -R2 on cell surface. Treatment of proteasome inhibitor bortezomib and histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) did not increase the cell surface expression of TRAIL-R1 and -R2 in myeloma cells. However, bortezomib and SAHA induced reduction of cellular FLICE inhibitory protein (c-FLIP) and synergistically enhanced the effect of R1-B12 but not of R2-E11 on apoptosis induction of TRAIL-resistant U266 cells. These results suggest that TRAIL-R1 mainly contribute to TRAIL-induced apoptosis in myeloma cells and that R1-B12 may have the therapeutic potential in combination with bortezomib or HDAC inhibitors in patients with multiple myeloma.

EGPred: prediction of eukaryotic genes using ab initio methods after combining with sequence similarity approaches.

EGPred is a Web-based server that combines ab initio methods and similarity searches to predict genes, particularly exon regions, with high accuracy. The EGPred program proceeds in the following steps: (1) an initial BLASTX search of genomic sequence against the RefSeq database is used to identify protein hits with an E-value <1; (2) a second BLASTX search of genomic sequence against the hits from the previous run with relaxed parameters (E-values <10) helps to retrieve all probable coding exon regions; (3) a BLASTN search of genomic sequence against the intron database is then used to detect probable intron regions; (4) the probable intron and exon regions are compared to filter/remove wrong exons; (5) the NNSPLICE program is then used to reassign splicing signal site positions in the remaining probable coding exons; and (6) finally ab initio predictions are combined with exons derived from the fifth step based on the relative strength of start/stop and splice signal sites as obtained from ab initio and similarity search. The combination method increases the exon level performance of five different ab initio programs by 4%-10% when evaluated on the HMR195 data set. Similar improvement is observed when ab initio programs are evaluated on the Burset/Guigo data set. Finally, EGPred is demonstrated on an approximately 95-Mbp fragment of human chromosome 13. The list of predicted genes from this analysis are available in the supplementary material. The EGPred program is computationally intensive due to multiple BLAST runs during each analysis. The EGPred server is available at http://www.imtech.res.in/raghava/egpred/.

In vivo excision and amplification of large human genomic segments using the Cre/loxP-and large T antigen/SV40 ori-mediated machinery

In vivo excision and amplification of pre-determined large genomic segments, directly from the genome of a natural host, can be a powerful tool for obtaining the genomic sequences with minimum rearrangements. In this study, an in vivo excision and amplification system in human BJAB cells was devised by combining the Cre/loxP system of bacteriophage P1 and the large T antigen/SV40 ori system of Simian virus 40. Two loxP sequences, each of which serves as a recognition site for recombinase Cre, were integrated unidirectionally into 5′- and 3′-untranslated regions (UTRs) of the human iNOS. An SV40 ori sequence, which serves as a conditional replication system, was inserted between the loxP sites. Trans-acting genes cre and large T antigen, which were under the control of a tetracycline responsive promoter, were also inserted into the 5′- and 3′-UTRs of the iNOS, respectively, by homologous recombination. Upon induction by doxycycline, the 45-kb iNOS genomic fragment of human chromosome 17 flanked by two loxP sites was excised and amplified up to about 45 copies per cell. Our method is very useful for obtaining large genomic fragments in quantities directly from human cells without using foreign hosts. Therefore, our approach can be used effectively for gap sequencing of a genome, gene therapy, and functional analysis of unknown genes in human cells.

In vivo excision and amplification of large human genomic segments using the Cre/ loxP-and large T antigen/SV40 ori-mediated machinery

In vivo excision and amplification of pre-determined large genomic segments, directly from the genome of a natural host, can be a powerful tool for obtaining the genomic sequences with minimum rearrangements. In this study, an in vivo excision and amplification system in human BJAB cells was devised by combining the Cre/ loxP system of bacteriophage P1 and the large T antigen/SV40 ori system of Simian virus 40. Two loxP sequences, each of which serves as a recognition site for recombinase Cre, were integrated unidirectionally into 5′- and 3′-untranslated regions (UTRs) of the human iNOS. An SV40 ori sequence, which serves as a conditional replication system, was inserted between the loxP sites. Trans-acting genes cre and large T antigen, which were under the control of a tetracycline responsive promoter, were also inserted into the 5′- and 3′-UTRs of the iNOS, respectively, by homologous recombination. Upon induction by doxycycline, the 45-kb iNOS genomic fragment of human chromosome 17 flanked by two loxP sites was excised and amplified up to about 45 copies per cell. Our method is very useful for obtaining large genomic fragments in quantities directly from human cells without using foreign hosts. Therefore, our approach can be used effectively for gap sequencing of a genome, gene therapy, and functional analysis of unknown genes in human cells.

Genomic DNA is captured and amplified during double-strand break (DSB) repair in human cells.

Genomic stability is maintained by the surveillance and repair of DNA damage. Here, we describe a mechanism whereby repair of extrachromosomal DNA double-strand breaks (DSBs) in human cells can be accompanied by capture of genomic DNA fragments. The availability of the human genome sequence enabled us to characterize these inserts in cells from a normal individual and from a patient with ataxia telangiectasia (AT), deficient for the damage response kinase ATM and prone to genomic instability. We find AT cells exhibit insertions of human chromosomal DNA fragments in excess of 17 kb during DSB repair, whereas we detected no such genomic inserts in normal cells. However, the presence of simian virus 40 (SV40), used to transform these cell lines, resulted in capture of genomic DNA associated with sites of viral integration in both cell types. The genomic instability at sites of SV40 integration was exported to other sites of DNA damage, and acquisition of the viral origin of replication resulted in gene amplification through autonomous replication of the plasmid harbouring the repaired extrachromosomal DSB. Should this same phenomenon apply to the repair of chromosomal DSBs, genome rearrangements made possible via this DSB insertional repair pose risks to genomic integrity, and may contribute to tumorigenic progression.