Repeat expansion by homologous recombination in the mouse germ line at palindromic sequences

Genetic instability can be induced by unusual DNA structures and sequence repeats. We have previously demonstrated that a large palindrome in the mouse germ line derived from transgene integration is extremely unstable and undergoes stabilizing rearrangements at high frequency, often through deletions that produce asymmetry. We have now characterized other palindrome rearrangements that arise from complex homologous recombination events. The structure of the recombinants is consistent with homologous recombination occurring by a noncrossover gene conversion mechanism in which a break induced in the palindrome promotes homologous strand invasion and repair synthesis, similar to mitotic break repair events reported in mammalian cells. Some of the homologous recombination events led to expansion in the size of the palindromic locus, which in the extreme case more than doubled the number of repeats. These results may have implications for instability observed at naturally occurring palindromic or quasipalindromic sequences.     

A system for assaying homologous recombination at the endogenous human thymidine kinase gene.

A system for assaying human interchromosomal recombination in vitro was developed, using a cell line containing two different mutant thymidine kinase genes (TK) on chromosomes 17. Heteroalleles were generated in the TK+/+ parent B-lymphoblast cell line WIL-2 by repeated exposure to the alkylating nitrogen mustard ICR-191, which preferentially causes +1 or -1 frameshifts. Resulting TK-/- mutants were selected in medium containing the toxic thymidine analog trifluorothymidine. Mutations were characterized by exon-specific polymerase chain reaction amplification and direct sequencing. In two lines, heterozygous frameshifts were located in exons 4 and 7 of the TK gene separated by approximately 8 kilobases. These lines undergo spontaneous reversion to TK+ at a frequency of less than 10(-7), and revertants can be selected in cytidine/hypoxanthine/aminopterin/thymidine medium. The nature and location of these heteroallelic mutations make large deletions, rearrangements, nondisjunction, and reduplication unlikely mechanisms for reversion to TK+. The mode of reversion to TK+ was specifically assessed by DNA sequencing, use of single-strand conformation polymorphisms, and analysis of various restriction fragment length polymorphisms (RFLPs) linked to the TK gene on chromosome 17. Our data suggest that a proportion of revertants has undergone recombination and gene conversion at the TK locus, with concomitant loss of frameshifts and allele loss at linked RFLPs. Models are presented for the origin of two recombinants.     

L1 sequences in HeLa extrachromosomal circular DNA: evidence for circularization by homologous recombination.

Subcloned probes of the L1 family of repetitive elements were used to isolate L1-carrying clones from a plasmid library of HeLa cell extrachromosomal circular DNA. One clone was analyzed in detail by restriction mapping, cross-hybridization to L1 probes, and base sequence analysis. In addition to approximately the 3' half of a full-sized L1 element, this clone carried 390 base pairs of non-L1 sequence that is single copy in the HeLa genome. A HeLa genomic clone of this unique chromosomal region was isolated and the sequence organization of the circle clone was compared with the linear chromosomal region from which it was ultimately derived. We discuss possible mechanisms of circular DNA formation and propose homologous intrachromosomal recombination between 9-base-pair direct repeats to be most likely in this case.     

Positive genetic selection for gene disruption in mammalian cells by homologous recombination.

Efficient modification of genes in mammalian cells by homologous recombination has not been possible because of the high frequency of nonhomologous recombination. An efficient method for targeted gene disruption has been developed. Cells with substitution of exogenous sequences into a chromosomal locus were enriched, by a factor of 100, using a positive genetic selection that specifically selects for homologous recombination at the targeted site. The selection is based on the conditional expression of a dominant selectable marker by virtue of in-frame gene fusion with the target gene. The dominant selectable marker was derived by modification of the Escherichia coli neo gene so that it retains significant activity in mammalian cells after in-frame fusion with heterologous coding sequences. In the example presented here, homologous recombinants were efficiently recovered from a pool in which the targeted gene was disrupted in 1 per 10,000 cells incorporating exogenous DNA.     

Intramolecular recombination between partially homologous sequences in Escherichia coli and Xenopus laevis oocytes.

We describe a system to analyze the individual contribution of a single physical DNA end on intramolecular recombination between partially homologous sequences. We took advantage of this partial sequence divergence to measure the distance separating the DNA end from the final recombination event. We show that a single physical DNA end stimulates recombination when located in a region of homology. Recombination frequency decreases gradually with the distance from the DNA end. A recombinational hot spot is found at the end of the region of homology. A large insertion of unrelated DNA interferes asymmetrically with this process, suggesting that a recombinogenic signal propagates along the region of homology.     

Molecular cloning of a human glycophorin B cDNA: nucleotide sequence and genomic relationship to glycophorin A.

Here we describe the isolation and nucleotide sequence of a human glycophorin B cDNA. The cDNA was identified by differential hybridization of synthetic oligonucleotide probes to a human erythroleukemic cell line (K562) cDNA library constructed in phage vector lambda gt10. The nucleotide sequence of the glycophorin B cDNA was compared with that of a previously cloned glycophorin A cDNA. The nucleotide sequences encoding the NH2-terminal leader peptide and first 26 amino acids of the two proteins are nearly identical. This homologous region is followed by areas specific to either glycophorin A or B and a number of small regions of homology, which in turn are followed by a very homologous region encoding the presumed membrane-spanning portion of the proteins. We used RNA blot hybridization with both cDNA and synthetic oligonucleotide probes to prove our previous hypothesis that glycophorin B is encoded by a single 0.5- to 0.6-kb mRNA and to show that glycophorins A and B are negatively and coordinately regulated by a tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate. We established the intron/exon structure of the glycophorin A and B genes by oligonucleotide mapping; the results suggest a complex evolution of the glycophorin genes.     

X/Y translocations resulting from recombination between homologous sequences on Xp and Yq.

Several regions of sequence homology between the human X and Y chromosomes have been identified. These segments are thought to represent areas of these chromosomes that have engaged in meiotic recombination in relatively recent evolutionary times. Normally, the X and Y chromosomes pair during meiosis and exchange DNA only within the pseudoautosomal region at the distal short arms of both chromosomes. However, it has been suggested that aberrant recombination involving other segments of high homology could be responsible for the production of X/Y translocations. We have studied four X/Y translocation patients using molecular probes detecting homologous sequences on X and Y chromosomes. In one translocation the breakpoints have been isolated and sequenced. The mapping data are consistent with the hypothesis that X/Y translocations arise by homologous recombination. The sequencing data from one translocation demonstrate this directly.     

A fundamental division in the Alu family of repeated sequences.

The Alu family of repeated sequences from the human genome contains two distinct subfamilies. This division is based on different base preferences in a number of diagnostic sequence positions. One subfamily of the sequences, referred to as the Alu-J subfamily, is very similar to 7SL DNA in these positions. The other subfamily, Alu-S, can be divided further into well-defined branches of sequences. These findings revise the previous picture of the Alu family and expose their complex evolutionary dynamics. They reveal sequence variations of potential importance for the proliferation of Alu repeats and relate them to their structural features. In addition, they open the possibility of using different types of Alu sequences as natural markers for studying genetic rearrangements in the genome.     

Human monoamine oxidase A and B genes exhibit identical exon-intron organization.

Monoamine oxidases A and B [MAOA and MAOB; amine:oxygen oxidoreductase (deaminating) (flavin-containing), EC 1.4.3.4] play important roles in the metabolism of neuroactive, vasoactive amines and the Parkinsonism-producing neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Human MAOA and MAOB genes isolated from X chromosome-specific libraries span at least 60 kilobases, consist of 15 exons, and exhibit identical exon-intron organization. Exon 12 codes for the covalent FAD-binding-site and is the most conserved exon; the MAOA and MAOB exon 12 products share 93.9% peptide identity. These results suggest that MAOA and MAOB are derived from duplication of a common ancestral gene and provide insight on the structural/functional relationship of the enzyme products.     

Sources and evolution of human Alu repeated sequences.

Alu repeated sequences arising in DNA of the human lineage during about the last 30 million years are closely similar to a modern consensus. Alu repeats arising at earlier times share correlated blocks of differences from the current consensus at diagnostic positions in the sequence. Using these 26 positions, we can recognize four subfamilies and the older ones are each successively closer to the 7SL sequence. It appears that there has existed a series of conserved genes that are the primary sources of the Alu repeat family, presumably through retroposition. These genes have probably replaced each other in overlapping relays during the evolution of primates.     

Inactivation of mouse alpha-globin gene by homologous recombination: mouse model of hemoglobin H disease

We have disrupted the 5' locus of the duplicated adult alpha-globin genes by gene targeting in the mouse embryonic stem cells and created mice with alpha-thalassemia syndromes. The heterozygous knockout mice (.alpha/alpha alpha) are asymptomatic like the silent carriers in humans whereas the homozygous knockout mice (.alpha/.alpha) show hemolytic anemia. Mice with three dysfunctional alpha-globin genes generated by breeding the 5' alpha-globin knockouts (.alpha/alpha alpha) and the deletion type alpha-thalassemia mice (../alpha alpha) produce severe hemoglobin H disease and they die in utero. These results indicate that the 5' alpha-globin gene is the predominant locus in mice, and suggest that it is even more dominant than its human homologue.     

Polymorphism and evolution of Alu sequences in the human low density lipoprotein receptor gene.

Two clusters of Alu sequences in the human low density lipoprotein (LDL) receptor gene have been analyzed in detail. One Alu cluster is present within the intron separating exons 15 and 16 of the gene and contains a polymorphic Pvu II site. The presence or absence of this site gives rise to two allelic fragments of 14 and 16.5 kilobases, respectively, in genomic Southern blots using cloned cDNA probes. This DNA polymorphic site is caused by a single adenine to guanine transition within an Alu repetitive element. The second cluster of Alu sequences is located in exon 18 of the LDL receptor gene. Southern blotting of primate DNAs suggests that this cluster became associated with the gene about 30 million years ago. Comparison of bovine DNA sequences, which lack this Alu cluster, with those of the human indicates that the Alu sequences inserted in exon 18 in two independent events.     

Introduction of a lacZ reporter gene into the mouse int-2 locus by homologous recombination.

We demonstrate that the frequency of gene targeting is unaffected by the length of nonhomologous DNA transferred to a target chromosomal sequence. A result of this finding is that a much wider spectrum of designed genomic alterations is now feasible. As a first application, we inserted a 5.4-kilobase cassette of nonhomologous DNA into the int-2 locus in mouse embryo-derived stem cells by gene targeting. The inserted DNA contained a lacZ gene positioned to create an in-frame fusion with the int-2 protein-coding region. Upon differentiation of these cells to embryoid bodies, the int-2-lacZ fusion faithfully reproduced the expression pattern of int-2 RNA. This ability to target reporter genes, such as lacZ, to specific mouse loci, combined with the ability to move the tagged gene into different mutant backgrounds, may provide an ideal approach for analyzing interactions among genes that participate in a developmental network.     

Stimulation of recombination between homologous sequences on plasmid DNA and chromosomal DNA in Escherichia coli by N-acetoxy-2-acetylaminofluorene.

A plasmid containing a wild-type lac operon and a tetracycline-resistance gene was covalently modified by N-acetoxy-2-acetylaminofluorene and used to transform two series of Lac- Escherichia coli cell types. Each set contained wild-type and repair-deficient mutants. One set of cells contained a lacY mutation and the other a deletion of the entire lac operon. Survival and mutagenesis of the plasmid were measured as a function of the N-acetoxy-2-acetylaminofluorene concentration. The results indicate that when no homologous sequences are present in the chromosomal DNA, mutations occur at a low frequency: at 10% survival the frequency was 1-2 X 10(-4) mutants per transformant. When homologous sequences, the lacY allele, are present in the chromosomal DNA, Lac- plasmids are found at a high frequency in a recA-dependent, lexA-independent fashion: at 10% survival the frequency was 5-10 X 10(-2) mutants per transformant. Southern blot analysis of the restriction enzyme profiles of the resulting plasmid and host-cell DNA sequences showed recombinational transfer of host sequences to the N-acetoxy-2-acetylamino-fluorene-treated plasmid had occurred. When the host chromosomes contained Lac+ homologous sequences no mutants were found, indicating that the results were not caused by error-prone recombination.     

Generation of hybrid human immunodeficiency virus by homologous recombination.

Human immunodeficiency virus (HIV) type 1, isolated from diverse sources, exhibits genomic diversity. The mechanisms by which the genomic diversity takes place in individuals exposed to multiple virus isolates is yet to be elucidated. Genetic variation, in general, might result from mutagenic events such as point mutations, rearrangements (insertions and deletions), and recombination. In an attempt to evaluate the process of genetic diversity, we designed experiments to analyze recombination between HIV DNAs by using DNA transfection in cell cultures. Here we report the successful recombination between truncated HIV proviral DNAs with an overlap homology of 53 base pairs that leads to the formation of viable hybrid virus. Recombination was also seen between exogenous DNA introduced into cells and homologous HIV sequences resident in the cells. These results indicate that recombination among various HIV isolates may play a significant role in the generation of genetic diversity of HIV. Further, the method used here enables the construction of hybrid HIV genomes to identify the viral determinants responsible for tropism, replication, and cytopathic effects.     

Detection of sequences homologous to human retroviral DNA in multiple sclerosis by gene amplification.

Twenty-one patients with multiple sclerosis, chronic progressive type, were examined for DNA sequences homologous to a human retrovirus. Genomic DNA from peripheral blood mononuclear cells was analyzed for the presence of homologous sequences to the human T-cell leukemia/lymphoma virus type I (HTLV-I) long terminal repeat, 3' gag, pol, and env domains by the enzymatic in vitro gene amplification technique, polymerase chain reaction. Positive identification of homologous pol sequences was made in the amplified DNA from six of these patients (29%). Three of these six patients (14%) also tested positive for the env region, but not for the other regions tested. In contrast, none of the samples from 35 normal individuals studied was positive when amplified and tested with the same primers and probes. Comparison of patterns obtained from controls and from patients with adult T-cell leukemia or tropical spastic paraparesis suggests that the DNA sequences identified are exogenous to the human genome and may correspond to a human retroviral species. The data support the detection of a human retroviral agent in some patients with multiple sclerosis.     

Immunoglobulin differentiation is dictated by repeated recombination sequences within the V region prototype gene: A hypothesis

Analysis of the available DNA sequences of immunoglobulin light chain genes reveals a unique structural pattern. A stretch of about 15 nucleotides repeats five times within the variable (V) region gene, with few base changes. Identification of these homologous sequences is apparent in the embryonic V(lambda) gene and might also be recognized in V(kappa) genes isolated from a myeloma. Although different from each other, the V(lambda) and V(kappa) hyperhomologous sequences display a remarkable resemblance to different prokaryote sequences associated with recombinational events. The homologous sequences appear at all three sites where hypervariable regions of the mature peptide are encoded. In addition, they are located at the site where V/constant (C) recombination is supposed to take place. Consequently, a general model is proposed for immunoglobulin differentiation. The hyperhomologous loci are postulated to be comprised of recombination sequences which makes them available for a mechanism of single-stranded DNA exposure. B cell maturation begins with V/C recombination, a step that is rate limiting. The fidelity of the process is ensured by extensive DNA homology between the two embryonic subgenes of V and C. Next, an error-prone repair system is activated and thereby introduces changes into the content of the immunoglobulin gene at the exposed loci. The process ends when mutations make the recombination sequence unrecognizable as such. The model is consistent with large amounts of data and is compatible with the view that immunoglobulin diversity is being generated somatically.