Promoter suppression in cultured mammalian cells can be blocked by the chicken beta-globin chromatin insulator 5'HS4 and matrix/scaffold attachment regions.

Studies have indicated that two transcriptionally active units can repress one another when they lie adjacent in head-to-tail tandem on a chromosome. Repression of a downstream (3') unit by an upstream (5') unit is known as "transcriptional interference", whereas repression of a 5' unit by a 3' unit is termed "promoter suppression". These two processes can occur between head-to-tail tandem copies of a transgene, or between transgenes and adjacent chromosomal genes. Interference can be blocked by inserting a transcription terminator between adjacent units. Here, we report that "promoter suppression" could be blocked by the insulator 5' DNaseI hypersensitive site 4, or matrix/scaffold attachment regions (MAR/SARs), when these elements were interposed between adjacent units. Because intergenic spacers of many repeated eukaryotic genes contain MAR/SARs and insulators, our observations suggest that these elements have the ability to segregate repeated genes into domains that act independently of one another. Our observations also suggest strategies to design transgenes that can act as autonomous units of expression.

Effects of sequence divergence on the efficiency of extrachromosomal recombination in mismatch repair proficient and deficient mammalian cell lines.

We have examined the effect of sequence divergence on the efficiency of recombination in mismatch repair proficient and deficient cell lines by using an exon-switch based assay that involves introns as recombination substrates. Sequence divergence of 15% decreased spontaneous recombination by six-fold in mismatch repair proficient cells but only three- and two-fold in human cells with defects in mismatch repair genes MLH1 and MSH2, respectively. The decrease in recombination efficiency in mismatch repair proficient background does not seem to be due to the production of rearranged recombination intermediates since these were readily detectable in the assay system we used. In contrast, the efficiency of double-strand break-induced recombination was not affected by sequence divergence in mismatch repair proficient or deficient background. These results indicate that sequence divergence and mismatch repair block initiation of spontaneous recombination but not recombinational repair of double-strand breaks. The differential sensitivity of these two processes may be required for genome stability.

The processing of DNA ends at double-strand breaks during homologous recombination: different roles for the two ends.

We have investigated the role of DNA ends during gap repair by homologous recombination. Mouse cells were transfected with a gapped plasmid carrying distinctive ends: on one side mouse LINE-1 repetitive sequences (L1Md-A2), and on the other rat LINE-1 sequences (L1Rn-3). The gap could be repaired by homologous recombination with endogenous mouse genomic LINE-1 elements, which are on average 95% and 85% homologous to L1Md-A2 and L1Rn-3 ends, respectively. Both L1Md-A2 and L1Rn-3 ends were found to initiate gap repair with equal efficiency. However, there were two types of gap repair products--precise and imprecise--the occurrence of which appears to depend on which end had been used for initiation and thus which end was left available for subsequent steps in recombination. These results, together with sequence analysis of recombinants obtained with plasmids having either mouse or rat LINE-1 sequences flanking the gap, strongly suggest that the two DNA ends played different roles in recombinational gap repair. One end was used to initiate the gap repair process, while the other end was involved at later steps, in the resolution of the recombination event.

Ectopic gene targeting exhibits a bimodal distribution of integration in murine cells, indicating that both intra- and interchromosomal sites are accessible to the targeting vector.

Ectopic gene targeting is an alternative outcome of the gene targeting process in which the targeting vector acquires sequences from the genomic target but proceeds to integrate elsewhere in the genome. Using two-color fluorescent in situ hybridization analysis, we have determined the integration sites of the gene targeting vector with respect to the target locus in a murine fibroblast line (LTA). We found that for ectopic gene targeting the distribution of integration sites was bimodal, being either within 3 Mb of the target or on chromosomes distinct from the chromosome carrying the target locus. Inter- and intrachromosomal sites appeared to be equally accessible to the targeting vector, with site-specific variations. Interestingly, interphase analysis indicated that vector sequences which had integrated ectopically in chromosomes other than the target colocalized with the target locus at a significant frequency compared to that of colocalization to random unlinked loci. We propose that ectopic gene targeting could be used to determine which chromosomal domains within the genome are accessible to a given genetic locus. Thus, recombination access mapping may present a new paradigm for the analysis of DNA accessibility and interaction within the genome.

Homologous junctions formed between a vector and human genomic repetitive LINE-1 elements as a result of one-sided invasion.

Studies on homologous recombination in mammalian cells between an exogenous DNA molecule containing a double-strand break and a homologous genomic sequence have indicated that there were at least two distinct types of homologous recombination processes, one that involved the formation of two homologous junctions and another that involved the formation of one homologous junction and one illegitimate junction. Both types of events are produced in gene targeting experiments. We have proposed a model to account for the later process called one-sided invasion. One-sided invasion has now been reported in numerous species belonging to different phyla and appears to be a universal mechanism. It has also been observed in normal human germ cells. The role of one-sided invasion is still unknown. Using a recombination assay between LINE-1 elements from the human genome and exogenous LINE-1 sequences, we have characterized the process of homologous junction formation in one-sided invasion. We found that at each of the homologous junctions, variable lengths of the vector L1 sequences had been replaced by genomic L1 sequences. We also found a homologous junction that involved three partners, suggesting that the homologous end could be released and become available for a second round of interaction.

Differential behavior of curved DNA upon untwisting.

We have synthesized DNA segments with different handedness, twisting and radii of curvature, and have analyzed the effect of untwisting on them. The results indicate that the dynamic behavior of curved DNA upon untwisting is strongly determined by the initial sequence-dependent DNA trajectory. In particular, DNA with the same radii but with opposite handedness of superhelix twisting can show very different conformational responses to ethidium bromide untwisting. Upon treatment with ethidium bromide, right-handed superhelixes decrease their twist and increase the planarity of the superhelix, while left-handed superhelixes increase twisting and decrease their degree of planarity.

Interference of DNA sequence divergence with precise recombinational DNA repair in mammalian cells.

Studies done in prokaryotes and eukaryotes have indicated that DNA sequence divergence decreases the frequency of homologous recombination. To determine which step(s) of homologous recombination is sensitive to DNA sequence divergence in mammalian cells we have used an assay that does not rely on the recovery of functional products. The assay is based on the acquisition by homologous recombination of endogenous LINE-1 sequences by exogenous LINE-1 sequences. In parallel experiments, we introduced into mouse cells two gapped exogenous LINE-1 sequences, one from the mouse, L1Md-A2, and the other from the rat, L1Rn-3. Although L1Rn-3 is on average less than 85% homologous to the LINE-1 elements of the mouse, the frequency of homologous recombination with endogenous LINE-1 elements obtained with L1Rn-3 was the same as the one obtained with L1Md-A2 which is on average 95% homologous to the LINE-1 elements of the mouse. The endogenous LINE-1 sequences rescued by L1Rn-3 were 8-18% divergent from L1Rn-3 sequences, whereas those rescued by L1Md-A2 were 2-5% divergent from L1Md-A2 sequences. The gap which had been introduced into the exogenous LINE-1 sequences had been precisely repaired in 50% of the recombinants obtained with L1Md-A2. None of the L1Rn-3 recombinants showed precise gap repair.(ABSTRACT TRUNCATED AT 250 WORDS)

Integration of a vector containing a repetitive LINE-1 element in the human genome.

Mammalian cells contain numerous nonallelic repeated sequences, such as multicopy genes, gene families, and repeated elements. One common feature of nonallelic repeated sequences is that they are homeologous (not perfectly identical). Our laboratory has been studying recombination between homeologous sequences by using LINE-1 (L1) elements as substrates. We showed previously that an exogenous L1 element could readily acquire endogenous L1 sequences by nonreciprocal homologous recombination. In the study presented here, we have investigated the propensity of exogenous L1 elements to be involved in a reciprocal process, namely, crossing-overs. This would result in the integration of the exogenous L1 element into an endogenous L1 element. Of over 400 distinct integration events analyzed, only 2% involved homologous recombination between exogenous and endogenous L1 elements. These homologous recombination events were imprecise, with the integrated vector being flanked by one homologous and one illegitimate junction. This type of structure is not consistent with classical crossing-overs that would result in two homologous junctions but rather is consistent with one-sided homologous recombination followed by illegitimate integration. Contrary to what has been found for reciprocal homologous integration, the degree of homology between the exogenous and endogenous L1 elements did not seem to play an important role in the choice of recombination partners. These results suggest that although exogenous and endogenous L1 elements are capable of homologous recombination, this seldom leads to crossing-overs. This observation could have implications for the stability of mammalian genomes.

Association of a host DNA structure with retroviral integration sites in chromosomal DNA.

Integration of retroviral genomes is a site-specific process with respect to the virus but not the host genome. Numerous chromosomal sites and various sequences can be used as targets. Nevertheless, preferential regions and integration patterns have been observed. Using a functional assay, we investigated if host structural DNA elements could be associated with retroviral integration sites. The results were that 9 of 10 distinct retroviral integration events occurred in close proximity of structural elements behaving like intrinsically bent DNA.

One-sided invasion events in homologous recombination at double-strand breaks.

Two classes of homologous recombination mechanism for repair of double-strand breaks (DSBs) have been described in eukaryotes so far. One is conservative and has been explained by the double-strand break repair model (Szostak et al., 1983), whereas the other one is non-conservative and has been explained by the single-strand annealing model (Lin et al., 1984). Here, we will review data supporting the existence of another homologous recombination mechanism for double-strand break repair. We will present the one-sided invasion model that we have proposed to explain this mechanism and discuss its potential implication in various homologous recombination events.

Chromosomal illegitimate recombination in mammalian cells is associated with intrinsically bent DNA elements.

Illegitimate recombination is the most frequent mechanism for chromosomal rearrangements in mammalian cells, yet little is known about this process. Most of the studies to date have looked at the sequences present at illegitimate junctions. These revealed the presence of recurrent DNA motifs, none of which was consistently found. We have undertaken to determine if intrinsic DNA structures such as bent DNA elements could be a major determinant in chromosomal illegitimate recombination. Using a two dimensional electrophoretic assay we found that eight out of eight junctions, resulting from various types of chromosomal rearrangements, had migration behaviour characteristic of DNA containing intrinsically bent DNA elements. In all cases, these occurred within one kilobase of the junctions, and in most cases could be found in both participating DNA segments. We also found that these bent DNA elements were present before the recombination event. When we analysed the frequency of intrinsically bent DNA elements in random chromosomal fragments, we found it to be about one per 11 kilobases. Thus these results suggest that bent DNA is associated with chromosomal illegitimate recombination.

Characterization of nonconservative homologous junctions in mammalian cells.

Homologous recombination in mammalian cells between extrachromosomal molecules, as well as between episomes and chromosomes, can be mediated by a nonconservative mechanism. It has been proposed that the key steps in this process are the generation (by double-strand cleavage) of overlapping homologous ends, the creation of complementary single-strand ends (either by strand-specific exonuclease degradation or by unwinding of the DNA helix), and finally the creation of heteroduplex DNA by the annealing of the single-strand ends. We have analyzed in detail the structure of nonconservative homologous junctions and determined the contribution of each end to the formation of the junction. We have also analyzed multiple descendants from single recombination events. Two types of junctions were found. The majority (90%) of the junctions were characterized by a single crossover site. These crossover sites were distributed randomly throughout the junction. The remaining 10% of the junctions had mosaic patterns of parental markers. Furthermore, in 9 of 10 cases, multiple descendants from a single recombination event were identical. Thus, it appears that in most cases few parental markers were involved in junction formation. This finding suggests that nonconservative homologous junctions are mediated mainly by short heteroduplexes of a few hundred base pairs or less. These results are discussed in terms of the current models of nonconservative homologous recombination.

Genetic exchange between endogenous and exogenous LINE-1 repetitive elements in mouse cells.

The repetitive LINE (L1) elements of the mouse, which are present at about 10(5) copies per genome and share over 80% of sequence homology, were examined for their ability to undergo genetic exchange with exogenous L1 sequences. The exogenous L1 sequences, carried by a shuttle vector, consisted of an internal fragment from L1Md-A2, a previously described member of the L1 family of the mouse. Using an assay that does not require the reconstitution of a selectable marker we found that this vector, in either circular or linear form, acquired DNA sequences from endogenous L1 elements at a frequency of 10(-3) to 10(-4) per rescued vector. Physical analysis of the acquired L1 sequences revealed that distinct endogenous L1 elements acted as donors and that different subfamilies participated. These results demonstrate that L1 elements are readily capable of genetic exchange. Apart from gene conversion events, the acquisition of L1 sequences outside the region of homology suggested that a second mechanism was also involved in the genetic exchange. A model which accounts for this mechanism is presented and its potential implication on the rearrangement of L1 elements is discussed.

Identification of Haemophilus influenzae type b by a monoclonal antibody coagglutination assay.

A coagglutination assay using monoclonal antibody is described for the identification of Haemophilus influenzae type b. An immunoglobulin G2a monoclonal antibody, Hb-2, directed against a serotype-specific outer membrane protein of H. influenzae type b was adsorbed to Staphylococcus aureus Cowan 1 cells. In a dot enzyme immunoassay, Hb-2 reacted with 453 of 455 H. influenzae type b isolates and did not react with H. influenzae of other serotypes, untypeable H. influenzae strains, or other bacterial species. The Hb-2 coagglutination assay was evaluated by testing 136 H. influenzae type b strains selected on the basis of multilocus enzyme genotypes, 5 strains of another serotype, and 94 untypeable H. influenzae strains. The specificity of the coagglutination assay was demonstrated by the inhibition of the reaction by free Hb-2 monoclonal antibodies. The coagglutination assay was as specific as the dot enzyme immunoassay and can be rapidly performed and easily interpreted.

A monoclonal antibody directed against a serotype-specific, outer-membrane protein of Haemophilus influenzae type b.

Monoclonal antibodies (Mabs) were produced against outer-membrane proteins (OMPs) of Haemophilus influenzae type b. The clones were screened by ELISA with outer-membrane preparations of H. influenzae type b and untypable strains as coating antigens. Antibodies directed against the proteins of mol. wt (10(3)) 43, 37 and 13 were identified by immunoblotting of SDS-PAGE patterns of OMPs. Proteolytic enzyme treatments of the OMPs resulted in reduction of Mab reactivity as measured by ELISA. Furthermore, the absence of reactivity of Mab Hb-2 with a preparation of lipopolysaccharide confirmed the protein nature of its corresponding epitope. Binding assays with live bacteria showed that Hb-2 reacted with a cell surface-exposed antigenic determinant. Mab Hb-2 was bactericidal in vitro in the presence of complement. The characterisation of Hb-2 (IgG2a) by Western immunoblotting analysis revealed that it was directed against the 37 X 10(3)-mol. wt OMP. In a dot-enzyme immunoassay, Hb-2 reacted specifically with 326 strains of H. influenzae type b. It did not cross-react with the other serotypes or untypable strains of H. influenzae or with other bacterial species. This is the first report of a monoclonal antibody identifying a serotype-specific surface-exposed OMP of H. influenzae type b.

Rapid diagnosis of severe Haemophilus influenzae serotype b infections by monoclonal antibody enzyme immunoassay for outer membrane proteins.

A highly sensitive and specific enzyme immunoassay (EIA) for the detection of Haemophilus influenzae serotype b antigens in body fluids and broth cultures was developed, with a polyclonal antibody directed against polyribose phosphate as the solid-phase reagent and a biotinylated monoclonal antibody directed against H. influenzae type b outer membrane protein as the liquid-phase reagent. H. influenzae type b antigens could be detected in broth cultures containing as little as 50 organisms per ml. The sensitivity and specificity of this system were compared with those of two commercial kits and counterimmunoelectrophoresis. The overall detection of H. influenzae type b antigens in clinical specimens collected from children infected with H. influenzae type b was as follows: with Phadebact, 86 and 86% in cerebrospinal fluid and urine specimens, respectively; with Bactigen, 86, 80, and 92%, with counterimmunoelectrophoresis, 78, 73, and 75%, and with biotin-avidin EIA, 100, 100, and 100% for cerebrospinal fluid, serum, and urine specimens, respectively. In the biotin-avidin EIA, no positive reactions were noted in specimens collected from patients infected with other bacteria or from patients without evidence of bacterial infection, whereas false-positive reactions were found by counterimmunoelectrophoresis and the commercial kits. These results suggest that this monoclonal antibody reacting with the outer membrane protein is more specific and sensitive than the conventional methods using polyclonal antisera for the detection of H. influenzae type b antigens during severe infections in children.