Recombination and selection at Brassica self-incompatibility loci

In Brassica species, self-incompatibility is controlled genetically by haplotypes involving two known genes, SLG and SRK, and possibly an as yet unknown gene controlling pollen incompatibility types. Alleles at the incompatibility loci are maintained by frequency-dependent selection, and diversity at SLG and SRK appears to be very ancient, with high diversity at silent and replacement sites, particularly in certain "hypervariable" portions of the genes. It is important to test whether recombination occurs in these genes before inferences about function of different parts of the genes can be made from patterns of diversity within their sequences. In addition, it has been suggested that, to maintain the relationship between alleles within a given S-haplotype, recombination is suppressed in the S-locus region. The high diversity makes many population genetic measures of recombination inapplicable. We have analyzed linkage disequilibrium within the SLG gene of two Brassica species, using published coding sequences. The results suggest that intragenic recombination has occurred in the evolutionary history of these alleles. This is supported by patterns of synonymous nucleotide diversity within both the SLG and SRK genes, and between domains of the SRK gene. Finally, clusters of linkage disequilibrium within the SLG gene suggest that hypervariable regions are under balancing selection, and are not merely regions of relaxed selective constraint.     

Design of hybrid metalloproteinases from Bacillus]

Possibility of using recombination mechanisms for the construction of hybrids between relative proteins containing highly homologous regions has been demonstrated. In order to design hybrid neutral proteases (NPr) NPr B. amyloliquefaciens--NPr B. brevis genes encoding these enzymes have been cloned into the same plasmid in tandem orientation with subsequent recombination between them. With the help of sequential inactivation we have managed to ensure efficient selection at intermediate stages as well as at the completion stage of construction when the desirable hybrid forms exhibited proteolytic activity. Constructions containing expressed genes of the hybrid neutral proteases NPr B. amyloliquefaciens--NPr B. brevis were obtained. The presence of several regions of high homology between the genes of the two Bacilli neutral proteinases determines the possibility of obtaining of various variants of hybrid proteins with different properties.     

Specificity-determining regions of a lepidopteran-specific insecticidal protein produced by Bacillus thuringiensis

The lepidopteran-specific, insecticidal crystal proteins of Bacillus thuringiensis vary in toxicity to different species of lepidopteran larvae. We report studies of CryIA(a) and CryIA(c), two related proteins that have different degrees of toxicity to Heliothis virescens yet very similar degrees of toxicity to Manduca sexta. The amino acid differences between these proteins are located primarily between residues 280 and 722. We have constructed a series of chimeric proteins and determined their toxicities to both insects. The most significant findings arise from the replacement of three segments of the cryIA(c) gene with homologous portions of the cryIA(a) gene: codons 332-428, 429-447, and 448-722. Each of these segments contributed substantially and largely additively toward efficacy for H. virescens. However, replacement of the 429-447 segment of cryIA(c) gene with the cryIA(a) sequence resulted in a 27-50-fold reduction in toxicity toward M. sexta whereas the reduction in toxicity to H. virescens was only 3-4-fold. Subdivision of the 429-447 segment and replacements involving residues within this segment reduced toxicity to M. sexta by 5- to more than 2000-fold whereas toxicity to H. virescens was only reduced 3-10-fold. These observations indicate that: 1) different but overlapping regions of the cryIA(c) gene determine specificity to each of the two test insects; 2) some of the examined gene segments interact in determining specificity; and 3) different sequences in the cryIA(a) and cryIA(c) genes are required for maximal toxicity to M. sexta.     

Generation of hybrid genes and proteins by vaccinia virus-mediated recombination: application to human immunodeficiency virus type 1 env.

The ability of poxviruses to undergo intramolecular recombination within tandemly arranged homologous sequences can be used to generate chimeric genes and proteins. Genes containing regions of nucleotide homology will recombine to yield a single sequence composed of portions of both original genes. A recombinant virus containing two genes with a number of conserved regions will yield a population of recombinant viruses containing a spectrum of hybrid sequences derived by recombination between the original genes. This scheme has been used to generate hybrid human immunodeficiency virus type 1 env genes. Recombinant vaccinia viruses that contain two divergent env genes in tandem array have been constructed. In the absence of selective pressure to maintain both genes, recombination between conserved homologous regions in these genes generated a wide range of progeny, each of which expressed a novel variant polypeptide encoded by the newly created hybrid env gene. Poxvirus-mediated recombination may be applied to map type-specific epitopes, to create novel pharmaceuticals such as hybrid interferons, to study receptor-binding or enzyme substrate specificities, or to mimic the antigenic diversity found in numerous pathogens.     

A Translation Fusion Product of Two Different Insecticidal Crystal Protein Genes of Bacillus thuringiensis Exhibits an Enlarged Insecticidal Spectrum

Two truncated Bacillus thuringiensis crystal protein genes, belonging to the classes cryIA(b) and cryIC and both coding for insecticidal N-terminal fragments of the corresponding crystal proteins, were translationally fused. Expression of the gene fusion in Escherichia coli showed a biologically active protein with a toxicity spectrum that overlapped those of both contributing crystal proteins.     

Identification of novel cry-type genes from Bacillus thuringiensis strains on the basis of restriction fragment length polymorphism of the PCR-amplified DNA.

Two pairs of universal oligonucleotide primers were designed to probe the most conserved regions of all known cryI-type gene sequences so that the amplified PCR fragments of the DNA template from Bacillus thuringiensis strains may contain all possible cryI-type gene sequences. The restriction fragment length polymorphism (RFLP) patterns of the PCR-amplified fragments revealed that 14 distinct cry-type genes have been identified from 20 B. thuringiensis strains. Those cry-type genes included cryIA(a), cryIA(a), cryIA(b), cryIA(b), cryIA(c), cryIB, cryIC, cryIC, cryIC(b), cryID, cryIE, cryIF, cryIF, and cryIII (a dagger at the end of a gene designation indicates a novel cry-type gene determined by restriction mapping or DNA sequences). Among them, the sequences of cryIA(a), cryIA(b), cryIB, cryIC, cryIF, and cryIII were found to be different from the corresponding published cry gene sequences. Interestingly, five cry-type genes [cryIA(a)-, cryIB-, cryIC-, cryIC(b)-, and cryIF-type genes] and seven cry-type genes [cryIA(a)-, cryIA(b)-, cryIB-, cryIC-, cryIC(b)-, cryIF-, and cryIII-type genes] have been detected from B. thuringiensis subsp. morrisoni HD-12 and B. thuringiensis subsp. wuhanensis, respectively. Therefore, the PCR-RFLP typing system is a facile method to detect both known and novel cry genes existing in B. thuringiensis strains.     

Specificity domain localization of Bacillus thuringiensis insecticidal toxins is highly dependent on the bioassay system

The Bacillus thuringiensis cryIA(a) and cryIA(c) gene specificity regions were probed by creating and testing hybrid toxins both in vivo and in vitro against cultured insect cells or dissociated midgut epithelial cells. Toxin threshold dose determinations revealed that CryIA(c) is highly active against cultured Choristoneure fumiterana cells (CF-1) whereas CryIA(a) is nontoxic. In live insect bioassays, a reversed order of toxicity was observed. Hybrid analysis reversed that the CryIA(c) toxicity-determining region is located between codons 258 and 510. Two smaller subsections of this region (residues 258–358 and 450–510) were able to confer toxicity, although at lower levels, and one region (358–450) was present where progressive substitutions of CryIA(a) with cryIA(c) sequences had no effect. Exchanging the non-homologous N-terminal regions of CryIA(c) with CryIE suggested that the W-terminus does not play a role in specificity. One hybrid clone, MP80, displays a 99.3% homology to CryIA(b) but shows an 800-fold increase in toxicity to CF–1 cells relative to that shown by CryIA(b). Direct comparison between live Bombyx mori bioassays and a newly developed in vitro lawn assay using dissociated midgut epithelial cells from the same insect revealed striking differences in toxicity. The toxicity-determining region for B. mori larvae was determined to be between codons 283 and 450, although the 450–620 codon region may exert an influence on toxicity. In general, native or hybrid toxins showing little or no insect intoxication were very active against the epithelial cells, suggesting that factors other than toxin amino acid sequence play an important role in determining toxin specificity.     

Comparative analysis of sequences at the 5' end of the human and mouse apolipoprotein B genes

A comparison was made between the DNA sequences in two regions of the mouse and the human apolipoprotein B genes: the 5'-flanking sequence and the region between the first exon and the second intron. Considerable homology was observed, particularly in the immediate 5' region and in the second intron. Because promoter and enhancer elements have been previously localized to these regions in the human apolipoprotein B gene, it is proposed that regions of conserved base sequence delineate binding regions for regulatory proteins. In some cases, contiguous regions of homology are longer than expected for regions designed as recognition sites for individual nuclear proteins, and may define regions recognizable by a cluster of interacting proteins. Both the human and mouse genes contain repetitive elements and a hypervariable dinucleotide repeat.     

Transposon-encoded site-specific recombination: nature of the Tn3 DNA sequences which constitute the recombination site res.

The tnpR gene of transposon Tn3 encodes a site-specific recombination enzyme that acts at res, a DNA region adjacent to tnpR, to convert co-integrate intermediates of interreplicon transposition to the normal transposition end-products. We have used two complementary approaches to study the nature of the Tn3 recombination region, res. Firstly, the DNA-binding sites for tnpR protein were determined in DNase I protection experiments. These identified a 120-bp region between the tnpA and tnpR genes that can be subdivided into three separate protein-binding sites. Genetic dissection experiments indicate that few, if any, other sequences in addition to this 120-bp region are required for res function. Moreover, we have shown that the two directly repeated res regions within a molecule are unequal partners in the recombination reaction: a truncated res region, which is unable to recombine with a second identical res region, can recombine efficiently with an intact res region. This demonstration, along with the observation that tnpR/res recombination acts efficiently on directly repeated res regions within a molecule but inefficiently both on inverted res regions in the same molecule and in the fusion reaction between res regions in different molecules, leads us to propose that one-dimensional diffusion (tracking) of tnpR protein along DNA is used to locate an initial res region, and then to bring a second directly repeated res region into a position that allows recombination between the res regions.     

Application of the hypervariable region of the 16S rDNA sequence as an index for the rapid identification of species in the genus Alicyclobacillus

A comparison of the 16S rRNA gene (rDNA) sequences of seven type strains belonging to different Alicyclobacillus species (i.e., five validated species, one proposed species and one genomic species) suggested that the 5' end hypervariable region (259-273 bases in length) of 16S rDNA was specific for the respective type strains. Further phylogenetic analysis based on DNA sequences of the hypervariable region using 24 Alicyclobacillus strains revealed that the strains could be categorized into five species and the A. acidocaldarius-Alicyclobacillus genomic species 1 group. The hypervariable region was highly conserved among the five species: A. acidiphilus, A. acidoterrestris, A. cycloheptanicus, A. herbarius, and A. hesperidum. The strains in the A. acidocaldarius-Alicyclobacillus genomic species 1 group were subdivided into two clusters (Clusters I and II) based on DNA sequences in the hypervariable region. On the basis of phenotypic characteristics, chemotaxonomic and phylogenetic analyses, and DNA-DNA hybridization data, strains in Cluster I were grouped as Alicyclobacillus genomic species 1 and strains in Cluster II were re-identified as A. acidocaldarius, thereby demonstrating that the hypervariable regions were also highly conserved within these two species. These results suggest that as is the case with Bacillus, the hypervariable region is significantly species-specific in the genus Alicyclobacillus to distinguish Alicyclobacillus species by DNA sequence comparison of the hypervariable region.     

Domain III substitution in Bacillus thuringiensis delta-endotoxin CryIA(b) results in superior toxicity for Spodoptera exigua and altered membrane protein recognition.

To test our hypothesis that substitution of domain III of Bacillus thuringiensis delta-endotoxin (Cry) proteins might improve toxicity to pest insects, e.g., Spodoptera exigua, in vivo recombination was used to produce a number of cryIA(b)-cryIC hybrid genes. A rapid screening assay was subsequently exploited to select hybrid genes encoding soluble protoxins. Screening of 120 recombinants yielded two different hybrid genes encoding soluble proteins with domains I and II of CryIA(b) and domain III of CryIC. These proteins differed by only one amino acid residue. Both hybrid protoxins gave a protease-resistant toxin upon in vitro activation by trypsin. Bioassays showed that one of these CryIA(b)-CryIC hybrid proteins (H04) was highly toxic to S. exigua compared with the parental CryIA(b) protein and significantly more toxic than CryIC. In semiquantitative binding studies with biotin-labelled toxins and intact brush border membrane vesicles of S. exigua, this domain III substitution appeared not to affect binding-site specificity. However, binding to a 200-kDa protein by CryIA(b) in preparations of solubilized and blotted brush border membrane vesicle proteins was completely abolished by the domain III substitution. A reciprocal hybrid containing domains I and II of CryIC and domain III of CryIA(b) did bind to the 200-kDa protein, confirming that domain III of CryIA(b) was essential for this reaction. These results show that domain III of CryIC protein plays an important role in the level of toxicity to S. exigua, that substitution of domain III may be a powerful tool to increase the repertoire of available active toxins for pest insects, and that domain III is involved in binding to gut epithelium membrane proteins of S. exigua.     

Hybrid survival motor neuron genes in patients with autosomal recessive spinal muscular atrophy: new insights into molecular mechanisms responsible for the disease.

Spinal muscular atrophy (SMA) is a frequent autosomal recessive neurodegenerative disorder leading to weakness and atrophy of voluntary muscles. The survival motor-neuron gene (SMN), a strong candidate for SMA, is present in two highly homologous copies (telSMN and cenSMN) within the SMA region. Only five nucleotide differences within the region between intron 6 and exon 8 distinguish these homologues. Independent of the severity of the disease, 90%-98% of all SMA patients carry homozygous deletions in telSMN, affecting either exon 7 or both exons 7 and 8. We present the molecular analysis of 42 SMA patients who carry homozygous deletions of telSMN exon 7 but not of exon 8. The question arises whether in these cases the telSMN is truncated upstream of exon 8 or whether hybrid SMN genes exist that are composed of centromeric and telomeric sequences. By a simple PCR-based assay we demonstrate that in each case the remaining telSMN exon 8 is part of a hybrid SMN gene. Sequencing of cloned hybrid SMN genes from seven patients, as well as direct sequencing and single-strand conformation analysis of all patients, revealed the same composition in all but two patients: the base-pair differences in introns 6 and 7 and exon 7 are of centromeric origin whereas exon 8 is of telomeric origin. Nonetheless, haplotype analysis with polymorphic multicopy markers, Ag1-CA and C212, localized at the 5' end of the SMN genes suggests different mechanisms of occurrence, unequal rearrangements, and gene conversion involving both copies of the SMN genes. In approximately half of all patients, we identified a consensus haplotype, suggesting a common origin. Interestingly, we identified a putative recombination hot spot represented by recombination-stimulating elements (TGGGG and TGAGGT) in exon 8 that is homologous to the human deletion-hot spot consensus sequence in the immunoglobulin switch region, the alpha-globin cluster, and the polymerase alpha arrest sites. This may explain why independent hybrid SMN genes show identical sequences.     

Localization of functional domains in E. coli K-12 outer membrane porins.

The genes ompC and phoE of Escherichia coli K-12 encode outer membrane pore proteins that are very homologous. To study the structure-function relationship of these proteins, we have constructed a series of ompC-phoE hybrid genes in which the DNA encoding part of one protein is replaced by the corresponding part of the other gene. These hybrid genes were easily obtained by using in vivo recombination. The fusion sites in the hybrid genes were localized by restriction enzyme mapping. The hybrid gene products were normally expressed and they were characterized with respect to functions and properties in which the native OmpC and PhoE proteins differ, such as pore characteristics, the receptor activity for phages and the binding of specific antibodies. Three regions within the N-terminal 130 amino acids were localized which determine pore characteristics and a segment between residues 75 and 110 contains amino acids which determine specificity for PhoE phages. A major cell surface-exposed region is located between residues 142 and 267. This region contains residues which are required for the binding of monoclonal antibodies directed against the cell surface-exposed part of PhoE and residues which determine specificity for OmpC phages.     

Recombination and chimeragenesis by in vitro heteroduplex formation and in vivo repair.

We describe a simple method for creating libraries of chimeric DNA sequences derived from homologous parental sequences. A heteroduplex formed in vitro is used to transform bacterial cells where repair of regions of non-identity in the heteroduplex creates a library of new, recombined sequences composed of elements from each parent. Heteroduplex recombination provides a convenient addition to existing DNA recombination methods ('DNA shuffling') and should be particularly useful for recombining large genes or entire operons. This method can be used to create libraries of chimeric polynucleotides and proteins for directed evolution to improve their properties or to study structure-function relationships. We also describe a simple test system for evaluating the performance of DNA recombination methods in which recombination of genes encoding truncated green fluorescent protein (GFP) reconstructs the full-length gene and restores its characteristic fluorescence. Comprising seven truncated GFP constructs, this system can be used to evaluate the efficiency of recombination between mismatches separated by as few as 24 bp and as many as 463 bp. The optimized heteroduplex recombination protocol is quite efficient, generating nearly 30% fluorescent colonies for recombination between two genes containing stop codons 463 bp apart (compared to a theoretical limit of 50%).     

Intermolecular homologous recombination in plants.

To study DNA topological requirements for homologous recombination in plants, we have constructed pairs of plasmids that contain nonoverlapping deletions in the neomycin phosphotransferase gene [APH(3')II], which, when intact, confers kanamycin resistance to plant cells. Protoplasts isolated from Nicotiana tabacum were cotransformed with complementary pairs of plasmids containing these truncated gene constructs. Homologous recombination or gene conversion within the homologous sequences (6 to 405 base pairs) of the protein-coding region of the truncated genes led to the restoration of the functional APH(3')II gene, rendering these cells resistant to kanamycin. Circular plasmid DNAs recombined very inefficiently, independent of the length of the homologous region. A double-strand break in one molecule only slightly increased the recombination frequency. The most favorable substrates for recombination were linear molecules. In this case, the recombination frequency was positively correlated with the length of the homologous regions. The recombination frequency of plasmids linearized at sites proximal to the deletion-homology junction was significantly higher than when linearization was distal to the homologous region. Vector homology within cotransformed plasmid sequences also increased the recombination frequency.     

The recA-recBCD dependent recombination pathways of Serratia marcescens and Proteus mirabilis in Escherichia coli: functions of hybrid enzymes and hybrid pathways.

The physical maps of cloned recBCD gene regions of Serratia marcescens and Proteus mirabilis were correlated to genes located in this region. The genes thyA, recC, recB, recD and argA were organized as in Escherichia coli. The 3 rec genes code for the 3 different subunits of the RecBCD enzyme and produced enzymes promoting recombination and repair of UV damage in E coli. The recBCD-dependent stimulation of recombination at specific nucleotide sequences called Chi (Chi-activation) was determined in lambda red-gam-crosses. Chi-activation by the different RecBCD enzymes decreased in the order E coli greater than S marcescens greater than P mirabilis. In E coli cloned subunits genes from S marcescens and P mirabilis led to the formation of functional hybrid enzymes consisting of subunits from 2 or even 3 species. The origin of the RecC subunit present in the hybrid enzymes affected the degree of Chi-activation. Further, changes in Chi-activation occurred when the RecD subunit in the enzyme from E coli was replaced by RecD proteins from S marcescens or P mirabilis. This suggested that the RecD subunit determines not only whether or not Chi-activation is possible but also to which extent it occurs. Finally we have reconstituted recombination pathways of S marcescens and P mirabilis by combining the cloned recA and recBCD genes from these species in E coli deleted for recA and recBCD. Both pathways can efficiently promote recombination and repair. Studies are summarized which showed that levels of repair and recombination promoted by the recA-recBCD genes are mostly higher when the recA and recBCD genes came from the same species than from 2 different species (hybrid RecBCD recombination pathway). The data are interpreted to provide evidence that in vivo the RecA protein co-operates with the RecBCD enzyme in recombination and repair of UV damage.     

The C-terminal domain of pediocin-like antimicrobial peptides (class IIa bacteriocins) is involved in specific recognition of the C-terminal part of cognate immunity proteins and in determining the antimicrobial spectrum

The pediocin-like bacteriocins contain two domains: a cationic N-terminal beta-sheet domain that mediates binding of the bacteriocin to the target cell surface and a more hydrophobic C-terminal hairpin-like domain that penetrates into the hydrophobic part of the target cell membrane. The two domains are joined by a hinge, which enables movement of the domains relative to each other. In this study, 12 different hybrid bacteriocins were constructed by exchanging domains between 5 different bacteriocins. The hybrid bacteriocins were by and large highly potent (i.e. similar potencies as the parental bacteriocins) when constructed such that the recombination point was in the hinge region, indicating that the two domains function independently. The use of optimal recombination points was, however, crucial. Shifting the recombination point just one residue from the hinge could reduce the activity of the hybrid by 3-4 orders of magnitude. Most interestingly, the active hybrids displayed target cell specificities similar to those of the parental bacteriocin from which their membrane-penetrating C-terminal hairpin domain was derived. The results also indicate that the negatively charged aspartate reside in the hinge of most pediocin-like bacteriocins interacts with the C-terminal hairpin domain, perhaps by interacting with the positively charged residue that is present at one of the last three positions in the C-terminal end of most pediocin-like bacteriocins. Bacteria that produce pediocin-like bacteriocins also produce a cognate immunity protein that protects the producer from being killed by its own bacteriocin. Four different active hybrid immunity proteins constructed by exchanging regions between three different immunity proteins were tested for their ability to confer immunity to the hybrid bacteriocins. The results showed that the C-terminal half of the immunity proteins contains a region that directly or indirectly specifically recognizes the membrane-penetrating C-terminal hairpin domain of pediocin-like bacteriocins. The implications these results have on how pediocin-like bacteriocins and their immunity proteins interact with cellular specificity determinants (for instance a putative bacteriocin receptor) are discussed.     

Molecular cloning, genetic characterization and DNA sequence analysis of the recM region of Bacillus subtilis.

In Bacillus subtilis the recM gene, whose product is associated with DNA repair and recombination, has been located between the dnaX and rrnA genes. The recM gene has been cloned and analyzed. Analysis of the nucleotide sequence (3.741-kilobase) around recM revealed five open reading frames (orf). We have assigned recM and dnaX to two of this orf, given the gene order dnaX-orf107-recM-orf74-orf87. The organization of genes of the dnaX-orf107-recM region resembles the organization of genes in the dnaX-orf12-recR region of the Escherichia coli chromosome. Proteins of 24.2 and 17.0 kDa would result from translation of the wild type and in vitro truncated recM genes, and radioactive bands of proteins of molecular weights of 24.5 and 17.0 kDa were detected by the use of the T7promoter-expression system. The RecM protein contains a potential zinc finger domain for nucleic acid binding and a putative nucleotide binding sequence that is present in many proteins that bind and hydrolyze ATP. Strains, in which the recM gene has been insertionally inactivated, were generated and show a phenotype essentially the same as previously described recM mutants.     

Definition of a core region of RAG-2 that is functional in V(D)J recombination.

The products of the RAG-1 and RAG-2 genes cooperate to allow V(D)J recombination in lymphoid and non-lymphoid cells. As one step toward understanding the role of RAG-2, we have constructed mutated RAG-2 genes and examined their ability to support recombination of plasmid substrates in a fibroblast cell line. The mutations define essential and dispensable parts of the RAG-2 gene. Mutations in the N-terminal part eliminate almost all activity. In the central region of the protein, some but not all local alterations still allow recombination. On the other hand, proteins with large deletions from the C-terminal end, including one truncated by 25%, still retain activity, even though this part of the protein is highly conserved between species. Similar results were obtained with substrates that retain either a signal joint or a coding joint, or perform an inversion. Thus all basic features of V(D)J joining are retained in a RAG-2 protein with only the first 75% of the sequence.