Hypotheses for testing deviations from random integration: evidence for nonrandom retroviral integration

The use of a recently developed in vitro model for retroviral integration provides a means of statistically testing hypotheses concerning the distribution of integration sites and hypotheses about the sequence of proviral orientations. In this study, three null hypotheses are formulated and applied to previously published data. Statistical analyses of these data suggest that the distribution of integration sites may not be uniform, and the sequence of proviral orientations is not random. On the basis of these results and the observed clustering of orientations, it was postulated that if a DNA sequence was involved in nonrandom proviral integration, that sequence would be found in the regions where the orientations change direction with respect to the target DNA ("I" regions). Computer analyses for homologous and complementary DNA sequences were performed on all possible pairs of identifiable "I" regions. A common sequence (at least 8 bp in size) was found in three out of four regions and that sequence was absent elsewhere in the target DNA. A model, with features of recombination reminiscent of chi sequences in bacteria, is proposed that may account for these results.     

A novel common triplet profile for GC-rich prokaryotic genomes

It has been reported that there is a majority triplet profile among genomes, which was considered as a reflection of general mechanisms of genome evolution (Albrecht-Buehler, 2007). However, there are actually, according to our further analysis and at least among prokaryotic genomes, two common triplet profiles: one is from low-GC content genomes; the other is from high-GC content genomes. Both common profiles would be direct reflections of GC content variations and strand symmetry of genomic sequences.     

Chromosomal locations of members of a family of novel endogenous human retroviral genomes.

Human cellular DNA contains two distinguishable families of retroviral related sequences. One family shares extensive nucleotide sequence homology with infectious mammalian type C retroviral genomes (T. I. Bonner, C. O'Connell, and M. Cohen, Proc. Natl. Acad. Sci. USA 79:4709-4713, 1982; M. A. Martin, T. Bryan, S. Rasheed, and A. S. Khan, Proc. Natl. Acad. Sci. USA 78:4892-4896, 1981). The other family contains major regions of homology with the pol genes of infectious type A and B and avian type C and D retroviral genomes (R. Callahan, W. Drohan, S. Tronick, and J. Schlom, Proc. Natl. Acad. Sci. USA 79:5503-5507, 1982; I. M. Chiu, R. Callahan, S. R. Tronick, J. Schlom, and S. A. Aaronson, Science 223:364-370, 1984). Analysis of the human recombinant clone HLM-2 has shown that the pol gene in the latter family is located within an endogenous proviral genome (R. Callahan, I. M. Chiu, J. F. H. Wong, S. R. Tronick, B. A. Roe, S. A. Aaronson, and J. Schlom, Science 228:1208-1211, 1985). We show that the proviral genome in HLM-2 and the related recombinant clone HLM-25 are located, respectively, on human chromosomes 1 and 5. Other related proviral genomes are located on chromosomes 7, 8, 11, 14, and 17.     

Multiple groups of novel retroviral genomes in pigs and related species

In view of the concern over potential infection hazards in the use of porcine tissues and organs for xenotransplantation to humans, we investigated the diversity of porcine endogenous retrovirus (PERV) genomes in the DNA of domestic pigs and related species. In addition to the three known envelope subgroups of infectious gamma retroviruses (PERV-A, -B, and -C), classed together here as PERV group gamma 1, four novel groups of gamma retrovirus (gamma 2 to gamma 5) and four novel groups of beta retrovirus (beta 1 to beta 4) genomes were detected in pig DNA using generic and specific PCR primers. PCR quantification indicated that the retroviral genome copy number in the Landrace x Duroc F(1) hybrid pig ranged from 2 (beta 2 and gamma 5) to approximately 50 (gamma 1). The gamma 1, gamma 2, and beta 4 genomes were transcribed into RNA in adult kidney tissue. Apart from gamma 1, the retroviral genomes are not known to be infectious, and sequencing of a small number of amplified genome fragments revealed stop codons in putative open reading frames in several cases. Analysis of DNA from wild boar and other species of Old World pigs (Suidae) and New World peccaries (Tayassuidae) showed that one retrovirus group, beta 2, was common to all species tested, while the others were present among all Old World species but absent from New World species. The PERV-C subgroup of gamma1 genomes segregated among domestic pigs and were absent from two African species (red river hog and warthog). Thus domestic swine and their phylogenetic relatives harbor multiple groups of hitherto undescribed PERV genomes.     

Helicase-AID: A novel molecular device for base editing at random genomic loci

CRISPR-enabled deaminase base editing has become a powerful tool for precisely editing nucleotides on the chromosome. In this study DNA helicases, such as Escherichia coli DnaB, were fused to activation-induced cytidine deaminase (AID) to form enzyme complexes which randomly introduces edited bases throughout the chromosome. DnaB-AID was found to increase 2.5 × 10³ fold relative to the mutagenesis frequency of wildtype. 97.9% of these edits were observed on the leading strand during DNA replication suggesting deamination to be highly coordinated with DNA replication. Using DnaB-AID, a 371.4% increase in β-carotene production was obtained following four rounds of editing. In Saccharomyces cerevisiae Helicase-AID was constructed by fusing AID to one of the subunits of eukaryotic helicase Mcm2-7 complex, MCM5. Using MCM5-AID, the average editing efficiency of five strains was 2.1 ± 0.4 × 10³ fold higher than the native genomic mutation rate. MCM5-AID was able to improve β-carotene production of S. cerevisiae 4742crt by 75.4% following eight rounds of editing. The S. cerevisiae MCM5-AID technique is the first biological tool for generating and accumulating single base mutations in eukaryotic chromosomes. Since the helicase complex is highly conservative in all eukaryotes, Helicase-AID could be adapted for various applications and research in all eukaryotic cells.     

Compositional bimodality and evolution of retroviral genomes.

The compositional distributions of genomes, genes (and their third codon positions) and long terminal repeats from retroviruses of warm-blooded vertebrates are characterized by a striking bimodality which is accompanied by a remarkable compositional homogeneity within each retroviral genome. A first, major class of retroviral genomes is GC-rich, whereas a second, minor class is GC-poor. Representative expressed viral genomes from the two classes integrate in GC-rich and GC-poor isochores, respectively, of host genomes. The first class comprises all oncoviruses (except B-types and some D-types), the second, lentiviruses, spumaviruses, as well as B-type and some D-type oncoviruses (e.g., mouse mammary tumor virus and simian retroviruses type D, respectively). The compositional bimodal distribution of retroviral genomes and the accompanying compositional homogeneity within each retroviral genome appear to be the result of the compositional evolution of retroviral genomes in their integrated form.     

A random model approach to mapping quantitative trait loci for complex binary traits in outbred populations.

Mapping quantitative trait loci (QTL) for complex binary traits is more challenging than for normally distributed traits due to the nonlinear relationship between the observed phenotype and unobservable genetic effects, especially when the mapping population contains multiple outbred families. Because the number of alleles of a QTL depends on the number of founders in an outbred population, it is more appropriate to treat the effect of each allele as a random variable so that a single variance rather than individual allelic effects is estimated and tested. Such a method is called the random model approach. In this study, we develop the random model approach of QTL mapping for binary traits in outbred populations. An EM-algorithm with a Fisher-scoring algorithm embedded in each E-step is adopted here to estimate the genetic variances. A simple Monte Carlo integration technique is used here to calculate the likelihood-ratio test statistic. For the first time we show that QTL of complex binary traits in an outbred population can be scanned along a chromosome for their positions, estimated for their explained variances, and tested for their statistical significance. Application of the method is illustrated using a set of simulated data.     

In vivo selection of randomly mutated retroviral genomes.

Darwinian evolution, that is the outgrowth of the fittest variants in a population, usually applies to living organisms over long periods of time. Recently, in vitro selection/amplification techniques have been developed that allow for the rapid evolution of functionally active nucleic acids from a pool of randomized sequences. We now describe a modification of the nucleic acid-evolution protocol in which selection and amplification take place inside living cells by means of a retroviral-based replication system. We have generated a library of HIV-1 DNA genomes with random sequences in particular domains of the TAR element, which is the binding site for the Tat trans-activator protein. This mixture of HIV genomes was transfected into T cells and outgrowth of the fittest viruses was observed within two weeks of viral replication. The results of this in vivo selection analysis are consistent with the notion that primary sequence elements in both TAR bulge and loop domains are critical for Tat-mediated trans-activation and viral replication.     

A novel approach to identifying regulatory motifs in distantly related genomes

Although proven successful in the identification of regulatory motifs, phylogenetic footprinting methods still show some shortcomings. To assess these difficulties, most apparent when applying phylogenetic footprinting to distantly related organisms, we developed a two-step procedure that combines the advantages of sequence alignment and motif detection approaches. The results on well-studied benchmark datasets indicate that the presented method outperforms other methods when the sequences become either too long or too heterogeneous in size.     

A high throughput method for genome-wide analysis of retroviral integration

Retroviral and lentiviral vectors integrate their DNA into the host cell genome leading to stable transgene expression. Integration preferentially occurs in the proximity of active genes, and may in some case disturb their activity, with adverse toxic consequences. To efficiently analyze high numbers of lentiviral insertion sites in the DNA of transduced cells, we developed an improved high-throughput method called vector integration tag analysis (VITA). VITA is based on the identification of Genomic Tags associated to the insertion sites, which are used as signatures of the integration events. We use the capacity of MmeI to cleave DNA at a defined distance of its recognition site, in order to generate 21 bp long tags from libraries of junction fragments between vector and cellular DNA. The length of the tags is sufficient in most cases, to identify without ambiguity an unique position in the human genome. Concatenation, cloning and sequencing of the tags allow to obtain information about 20–25 insertion sites in a single sequencing reaction. As a validation of this method, we have characterized 1349 different lentiviral vector insertion sites in transduced HeLa cells, from only 487 sequencing reactions, with a background of <2% false positive tags.     

Transforming activity of retroviral genomes encoding Gag-Axl fusion proteins.

Retroviral genomes encoding a portion of the Moloney murine leukemia virus Gag protein fused to portions of the murine axl cDNA were constructed so as to mimic naturally occurring transforming viruses. Virus MA1 retained 5 amino acids of the extracellular domain and the complete transmembrane and intracellular domains of Axl; virus MA2 retained only the intracellular Axl sequences beginning 33 amino acids downstream of the transmembrane region. Although both viruses could transform NIH 3T3 cells, they induced different morphological changes. MA1 transformants became elongated and assumed a cross-hatched pattern, while MA2 transformants were round and very refractile and grew to high density. Gag-Axl and Glyco-Gag-Axl proteins were detected in both types of transformed cells and were predominantly localized to the cytoplasmic compartment. When cell-free v-axl virus supernatants were introduced into wild-type BALB/c neonates, Rag-2-deficient mice, or c-myc transgenic mice, they did not cause tumors in a 3-month period. However, MA2-transformed NIH 3T3 cells, but not MA1 or control cells, could establish sarcomas by subcutaneous or intraperitoneal injection into BALB/c neonates. These results show that the transforming potential of the axl gene can be activated by truncation of the extracellular domain of the receptor and fusion of the remaining sequence to the gag gene.     

PEX genes in fungal genomes: common, rare or redundant

PEX genes encode proteins, termed peroxins, that are required for the biogenesis and proliferation of microbodies (peroxisomes). We have screened the available protein and DNA databases to identify putative peroxin orthologs in 17 fungal species (yeast and filamentous fungi) and in humans. This analysis demonstrated that most peroxins are present in all fungi under study. Only Pex16p is absent in most yeast species, with the exception of Yarrowia lipolytica, but this peroxin is present in all filamentous fungi. Furthermore, we found that the Y. lipolytica PEX9 gene, a putative orphan gene, might encode a Pex26p ortholog. In addition, in the genomes of Saccharomyces cerevisiae and Candida glabrata, several PEX genes appear to have been duplicated, exemplified by the presence of paralogs of the peroxins Pex5p and Pex21p, which were absent in other organisms. In all organisms, we observed multiple paralogs of the peroxins involved in organelle proliferation. These proteins belong to two groups of peroxins that we propose to designate the Pex11p and Pex23p families. This redundancy may complicate future studies on peroxisome biogenesis and proliferation in fungal species.     

A compression-based approach for coding sequences identification. I. Application to prokaryotic genomes.

Most of the gene prediction algorithms for prokaryotes are based on Hidden Markov Models or similar machine-learning approaches, which imply the optimization of a high number of parameters. The present paper presents a novel method for the classification of coding and non-coding regions in prokaryotic genomes, based on a suitably defined compression index of a DNA sequence. The main features of this new method are the non-parametric logic and the costruction of a dictionary of words extracted from the sequences. These dictionaries can be very useful to perform further analyses on the genomic sequences themselves. The proposed approach has been applied on some prokaryotic complete genomes, obtaining optimal scores of correctly recognized coding and non-coding regions. Several false-positive and false-negative cases have been investigated in detail, which have revealed that this approach can fail in the presence of highly structured coding regions (e.g., genes coding for modular proteins) or quasi-random non-coding regions (e.g., regions hosting non-functional fragments of copies of functional genes; regions hosting promoters or other protein-binding sequences). We perform an overall comparison with other gene-finder software, since at this step we are not interested in building another gene-finder system, but only in exploring the possibility of the suggested approach.