Clusters of resistance genes in plants evolve by divergent selection and a birth-and-death process

Classical genetic and molecular data show that genes determining disease resistance in plants are frequently clustered in the genome. Genes for resistance (R genes) to diverse pathogens cloned from several species encode proteins that have motifs in common. These motifs indicate that R genes are part of signal-transduction systems. Most of these R genes encode a leucine-rich repeat (LRR) region. Sequences encoding putative solvent-exposed residues in this region are hypervariable and have elevated ratios of nonsynonymous to synonymous substitutions; this suggests that they have evolved to detect variation in pathogen-derived ligands. Generation of new resistance specificities previously had been thought to involve frequent unequal crossing-over and gene conversions. However, comparisons between resistance haplotypes reveal that orthologs are more similar than paralogs implying a low rate of sequence homogenization from unequal crossing-over and gene conversion. We propose a new model adapted and expanded from one proposed for the evolution of vertebrate major histocompatibility complex and immunoglobulin gene families. Our model emphasizes divergent selection acting on arrays of solvent-exposed residues in the LRR resulting in evolution of individual R genes within a haplotype. Intergenic unequal crossing-over and gene conversions are important but are not the primary mechanisms generating variation.     

The root knot nematode resistance gene Mi from tomato is a member of the leucine zipper, nucleotide binding, leucine-rich repeat family of plant genes

The Mi locus of tomato confers resistance to root knot nematodes. Tomato DNA spanning the locus was isolated as bacterial artificial chromosome clones, and 52 kb of contiguous DNA was sequenced. Three open reading frames were identified with similarity to cloned plant disease resistance genes. Two of them, Mi-1.1 and Mi-1.2, appear to be intact genes; the third is a pseudogene. A 4-kb mRNA hybridizing with these genes is present in tomato roots. Complementation studies using cloned copies of Mi-1.1 and Mi-1.2 indicated that Mi-1.2, but not Mi-1.1, is sufficient to confer resistance to a susceptible tomato line with the progeny of transformants segregating for resistance. The cloned gene most similar to Mi-1.2 is Prf, a tomato gene required for resistance to Pseudomonas syringae. Prf and Mi-1.2 share several structural motifs, including a nucleotide binding site and a leucine-rich repeat region, that are characteristic of a family of plant proteins, including several that are required for resistance against viruses, bacteria, fungi, and now, nematodes.     

Shortening of the symptom-free period in rhesus macaques is associated with decreasing nonsynonymous variation in the env variable regions of simian immunodeficiency virus SIVsm during passage

During six blood passages of simian immunodeficiency virus SIVsm in rhesus macaques, the asymptomatic period shortened from 18 months to 1 month. To study SIVsm envelope gene (env) evolution during passage in rhesus macaques, the C1 to CD4 binding regions of multiple clones were sequenced at seroconversion and again at death. The env variation found during adaptation was almost completely confined to the variable regions. Intrasample sequence variation among clones at seroconversion was lower than the variation among clones at death. Intrasample variation among clones from a single time point as well as intersample variation decreased during the passage. In the variable regions, the mean number of intrasample nonsynonymous nucleotide substitutions decreased from the first passage (5.26 x 10(-2) +/- 0.6 x 10(-2) per site) to the fifth passage (2.24 x 10(-2) +/- 0.4 x 10(-2) per site), whereas in the constant regions, the mean number of intrasample nonsynonymous nucleotide substitutions differed less between the first and fifth passages (1. 14 x 10(-2) +/- 0.27 x 10(-2) and 0.80 x 10(-2) +/- 0.24 x 10(-2) per site). Shortening of the asymptomatic period coincided with a rise in the Ks/Ka ratio (ratio between the number of synonymous [Ks] and the number of nonsynonymous [Ka] substitutions) from 1.080 in passage one to 1.428 in passage five and mimicked the difference seen in the intrahost evolution between asymptomatic and fast-progressing individuals infected with human immunodeficiency virus type 1. The distribution of nonsynonymous substitutions was biphasic, with most of the adaptation of env variable regions occurring in the first three passages. This phase, in which the symptom-free period fell to 4 months, was followed by a plateau phase of apparently reduced adaptation. Analysis of codon usage revealed decreased codon redundancy in the variable regions. Overall, the results suggested a biphasic pattern of adaptation and evolution, with extremely rapid selection in the first three passages followed by an equilibrium or stabilization of the variation between env clones at different time points in passages four to six.     

Isolation of NotI sites from chromosome 22q11

Chromosome 22q11 contains a large number of interesting loci, including genes associated with cancer and developmental defects. The region is also the site of the lambda immunoglobulin variable and constant regions and the BCR, gamma-glutamyl transpeptidase, and GGT-like activity multigene families. Because of the complexities associated with mapping highly related gene families, we have examined the utility of mapping large areas of DNA using a defined approach. A total of 21 complete NotI sites from band q11 were cloned and ordered into six noncontiguous clusters of sites using a combination of somatic cell hybrid panels, NotI jumping and linking libraries, and fluorescence in situ hybridization. The largest cluster spanned an estimated 2 Mb of NotI fragments, the smallest 115 kb. Approximately 3.5 Mb of band q11 could be examined for rearrangements in NotI restriction enzyme fragments. A number of conserved sequences, two genes, and a minimum of two families of related sequences were identified adjacent to NotI sites.     

Molecular evolution of an imprinted gene: repeatability of patterns of evolution within the mammalian insulin-like growth factor type II receptor

The repeatability of patterns of variation in Ka/Ks and Ks is expected if such patterns are the result of deterministic forces. We have contrasted the molecular evolution of the mammalian insulin-like growth factor type II receptor (Igf2r) in the mouse-rat comparison with that in the human-cow comparison. In so doing, we investigate explanations for both the evolution of genomic imprinting and for Ks variation (and hence putatively for mutation rate evolution). Previous analysis of Igf2r, in the mouse-rat comparison, found Ka/Ks patterns that were suggested to be contrary to those expected under the conflict theory of imprinting. We find that Ka/Ks variation is repeatable and hence confirm these patterns. However, we also find that the molecular evolution of Igf2r signal sequences suggests that positive selection, and hence conflict, may be affecting this region. The variation in Ks across Igf2r is also repeatable. To the best of our knowledge this is the first demonstration of such repeatability. We consider three explanations for the variation in Ks across the gene: (1) that it is the result of mutational biases, (2) that it is the result of selection on the mutation rate, and (3) that it is the product of selection on codon usage. Explanations 2 and 3 predict a Ka-Ks correlation, which is not found. Explanation 3 also predicts a negative correlation between codon bias and Ks, which is also not found. However, in support of explanation 1 we do find that in rodents the rate of silent C --> T mutations at CpG sites does covary with Ks, suggesting that methylation-induced mutational patterns can explain some of the variation in Ks. We find evidence to suggest that this CpG effect is due to both variation in CpG density, and to variation in the frequency with which CpGs mutate. Interestingly, however, a GC4 analysis shows no covariance with Ks, suggesting that to eliminate methyl-associated effects CpG rates themselves must be analyzed. These results suggest that, in contrast to previous studies of intragenic variation, Ks patterns are not simply caused by the same forces responsible for Ka/Ks correlations.     

The TRI11 gene of Fusarium sporotrichioides encodes a cytochrome P-450 monooxygenase required for C-15 hydroxylation in trichothecene biosynthesis

Several genes in the trichothecene biosynthetic pathway of Fusarium sporotrichioides have been shown to reside in a gene cluster. Sequence analysis of a cloned DNA fragment located 3.8 kb downstream from TRI5 has led to the identification of the TRI11 gene. The nucleotide sequence of TRI11 predicts a polypeptide of 492 residues (Mr = 55,579) with significant similarity to members of the cytochrome P-450 superfamily. TRI11 is most similar to several fungal cytochromes P-450 (23 to 27% identity) but is sufficiently distinct to define a new cytochrome P-450 gene family, designated CYP65A1. Disruption of TRI11 results in an altered trichothecene production phenotype characterized by the accumulation of isotrichodermin, a trichothecene pathway intermediate. The evidence suggests that TRI11 encodes a C-15 hydroxylase involved in trichothecene biosynthesis.     

Histamine H2 receptor-mediated airway goblet cell secretion and its modulation by histamine-degrading enzymes

The nucleotide sequences of seven Escherichia coli genes that encode members of the gluconate permease (GntP) family have recently become available. These genes include gntP, gntU, gntW, ORf449, dsdX, and ORFo454. The deduced amino acid sequences of all seven E. coli genes are homologous to the gntP gene products from Bacillus subtilis and B. licheniformis as well as two additional gene products from Haemophilus influenzae. These 11 proteins are not demonstrably homologous to members of the major facilitator superfamily or other recognized permease families. Four of the E. coli gluconate transporter genes have been cloned and shown to encode gluconate transporters with apparent affinities ranging from 6 to 212 microM. These studies serve to characterize a novel family of bacterial permeases.     

A walk in the chorion locus of Bombyx mori

We have recovered overlapping clones that represent in the aggregate a contiguous segment of chromosomal DNA 270 kb in length, or probably one third of the chorion locus of Bombyx mori. Approximately 70 genes have been identified, the majority of which are arranged in coordinately expressed pairs. The nonidentical genes expressed in the late period of choriogenesis are clustered within a single, 130 kb region, which is flanked by regions containing genes that are active during the middle developmental period. The late genes encode two families of high-cysteine proteins; the evolutionarily persistent clustering of these families contrasts sharply with the extensive sequence diversification of the structural genes and their flanking DNA elements. We discuss the possible regulatory significance of the clustered arrangement, as well as certain features of multigene family evolution.     

Type 2M von Willebrand disease: F606I and I662F mutations in the glycoprotein Ib binding domain selectively impair ristocetin- but not botrocetin-mediated binding of von Willebrand factor to platelets

von Willebrand disease (vWD) is a common, autosomally inherited, bleeding disorder caused by quantitative and/or qualitative deficiency of von Willebrand factor (vWF). We describe two families with a variant form of vWD where affected members of both families have borderline or low vWF antigen levels, normal vWF multimer patterns, disproportionately low ristocetin cofactor activity, and significant bleeding symptoms. Whereas ristocetin-induced binding of plasma vWF from affected members of both families to fixed platelets was reduced, botrocetin-induced platelet binding was normal. The sequencing of genomic DNA identified unique missense mutations in each family in the vWF exon 28. In Family A, a missense mutation at nucleotide 4105T --> A resulted in a Phe606Ile amino acid substitution (F606I) and in Family B, a missense mutation at nucleotide 4273A --> T resulted in an Ile662Phe amino acid substitution (I662F). Both mutations are within the large disulfide loop between Cys509 and Cys695 in the A1 domain that mediates vWF interaction with platelet glycoprotein Ib. Expression of recombinant vWF containing either F606I or I662F mutations resulted in mutant recombinant vWF with decreased ristocetin-induced platelet binding, but normal multimer structure, botrocetin-induced platelet binding, collagen binding, and binding to the conformation-sensitive monoclonal antibody, AvW-3. Both mutations are phenotypically distinct from the previously reported variant type 2MMilwaukee-1 because of the presence of normal botrocetin-induced platelet binding, collagen binding, and AvW-3 binding, as well as the greater frequency and intensity of clinical bleeding. When the reported type 2M mutations are mapped on the predicted three-dimensional structure of the A1 loop of vWF, the mutations cluster in one region that is distinct from the region in which the type 2B mutations cluster.     

Distribution of topoisomerase II-mediated cleavage sites and relation to structural and functional landmarks in 830 kb of Drosophila DNA

The pattern of sites for cleavage mediated by topoisomerase II was determined in 830 kb of cloned DNA from the Drosophila X chromosome, with the objectives of comparing it with mapped structural and functional landmarks and examining if the correlations with such landmarks reported in individual loci can be generalized to a region approximately 100 times longer. The relative frequencies of topoisomerase II cleavage sites in 247 restriction fragments from 67 clones were quantified by hybridization with probes prepared from DNA fragments which abutted all cleavage sites in each clone, selected through the covalently bound topoisomerase II subunit; the specificity and quantitative nature of this method were demonstrated using a plasmid DNA model. The 12 restriction fragments with strong nuclear scaffold attachment (SAR) activity, of which seven possess autonomous replication (ARS) activity, show statistically strong coincidence or contiguity ( P 10 kb; their sensitivity is therefore unlikely to be due to alternating purine-pyrimidine repeats or regions of Z conformation, which are preferred motifs. The hypothesis that they possess intrinsic curvature is consistent with the similarity of their length and spacing to regions of predicted curvature in the 315 kb DNA of Saccharomyces cerevisiae chromosome III and with the reported strong binding preference of topoisomerase II for curved DNA. The topoisomerase II cleavage pattern in this DNA further shows that its relationships to functional properties seen in individual loci, especially to MAR/SAR and ARS activity and to the restricted accessibility of DNA to topoisomerase II in vivo, can be generalized to much longer regions of the genome.     

Extreme structural heterogeneity among the members of a maize retrotransposon family

A few families of retrotransposons characterized by the presence of long terminal repeats (LTRs) have amplified relatively recently in maize and account for >50% of the genome. Surprisingly, none of these elements have been shown to cause a single mutation. In contrast, most of the retrotransposon-induced mutations isolated in maize are caused by the insertion of elements that are present in the genome at 2-50 copies. To begin to understand what limits the amplification of this mutagenic class of LTR-retrotransposons, we are focusing on five elements previously identified among 17 mutations of the maize waxy gene. One of these elements, Stonor, has sustained a deletion of the entire gag region and part of the protease domain. Missing sequences were recovered from larger members of the Stonor family and indicate that the deletion probably occurred during retrotransposition. These large elements have an exceptionally long leader of 2 kb that includes a highly variable region of approximately 1 kb that has not been seen in previously characterized retrotransposons. This region serves to distinguish each member of the Stonor family and indicates that no single element has yet evolved that can attain the very high copy numbers characteristic of other element families in maize.     

A gene cluster for 6-deoxy-L-talan synthesis in Actinobacillus actinomycetemcomitans

The serotype c antigen of Actinobacillus actinomycetemcomitans consists of 6-deoxy-l-talose. A gene cluster involved in the synthesis of serotype-specific polysaccharide antigen was cloned from the chromosomal DNA of A. actinomycetemcomitans NCTC 9710 (serotype c). This cluster consisted of 17 open reading frames. Escherichia coli produced the polysaccharide that reacts with the serotype c-specific antibody when transformed with a plasmid containing the cluster. Comparing the structure of the gene cluster with a similar cluster from A. actinomycetemcomitans Y4 (serotype b), which produces a polysaccharide consisting of l-rhamnose and d-fucose, revealed that a 5.7 kb region containing seven genes in the cluster from strain Y4 was replaced by a 3.8 kb region containing three genes in strain NCTC 9710. The results suggest that these region, as well as dTDP-6-deoxy-l-talose-forming dTDP-4-keto-l-rhamnose reductase, is essential to the production of extracellular polysaccharide specific to serotype c.     

The chlorocatechol-catabolic transposon Tn5707 of Alcaligenes eutrophus NH9, carrying a gene cluster highly homologous to that in the 1,2,4-trichlorobenzene-degrading bacterium Pseudomonas sp. strain P51, confers the ability to grow on 3-chlorobenzoate

Alcaligenes eutrophus (Ralstonia eutropha) NH9, isolated in Japan, utilizes 3-chlorobenzoate as its sole source of carbon and energy. Sequencing of the relevant region of plasmid pENH91 from strain NH9 revealed that the genes for the catabolic enzymes were homologous to the genes of the modified ortho-cleavage pathway. The genes from strain NH9 (cbnR-ABCD) showed the highest homology (89 to 100% identity at the nucleotide level) to the tcbR-CDEF genes on plasmid pP51 of the 1,2,4-trichlorobenzene-degrading bacterium Pseudomonas sp. strain P51, which was isolated in The Netherlands. The structure of the operon, including the lengths of open reading frames and intervening sequences, was completely conserved between the cbn and tcb genes. Most nucleotide substitutions were localized within and proximal to the cbnB (tcbD) gene. The difference in the chloroaromatics that the two strains could use as growth substrates seemed to be due to differences in enzymes that convert substrates to chlorocatechols. The restriction map of plasmid pENH91 was clearly different from that of pP51 except in the regions that contained the cbnR-ABCD and tcbR-CDEF genes, respectively, suggesting that the chlorocatechol gene clusters might have been transferred as units. Two homologous sequences, present as direct repeats in both flanking regions of the cbnR-ABCD genes on pENH91, were found to be identical insertion sequences (ISs), designated IS1600, which formed a composite transposon designated Tn5707. Although the tcbR-CDEF genes were not associated with similar ISs, a DNA fragment homologous to IS1600 was cloned from the chromosome of strain P51. The sequence of the fragment suggested that it might be a remnant of an IS. The two sequences, together with IS1326 and nmoT, formed a distinct cluster on a phylogenetic tree of the IS21 family. The diversity of the sources of these IS or IS-like elements suggests the prevalence of ISs of this type.     

The tomato Cf-5 disease resistance gene and six homologs show pronounced allelic variation in leucine-rich repeat copy number

The tomato Cf-2 and Cf-5 genes confer resistance to Cladosporium fulvum and map to a complex locus on chromosome 6. The Cf-5 gene has been isolated and is predicted to encode a largely extracytoplasmic protein containing 32 leucine-rich repeats (LRRs), resembling the previously isolated Cf-2 gene, which has 38 LRRs. Three haplotypes of this locus from Lycopersicon esculentum, L. pimpinellifolium, and L. esculentum var cerasiforme were compared, and five additional homologs of Cf-5 were sequenced. All share extensive sequence identity, particularly within the C-terminal portions of the predicted proteins. In striking contrast to the Cf-9 gene family, six of seven homologs in the Cf-2/Cf-5 gene family vary in LRR copy number, ranging from 25 to 38 LRRs. Cf-5 and one adjacent homolog differ by only two LRRs. Recombination events that vary the LRR copy number in this region could provide a mechanism for the generation of new specificities for recognition of different ligands. A recombination breakpoint between the Cf-2 and Cf-5 loci was fully characterized and shown to be intragenic.     

Complex beta-satellite repeat structures and the expansion of the zinc finger gene cluster in 19p12

We investigated the organization, architecture, and evolution of the largest cluster ( approximately 4 Mb) of Krüppel-associated box zinc finger (KRAB-ZNF) genes located in cytogenetic band interval 19p12. A highly integrated physical map ( approximately 700 kb) of overlapping cosmid and BAC clones was developed between genetic STS markers D19S454 and D19S269. Using ZNF91 exon-specific probes to interrogate a detailed EcoRI restriction map of the region, ZNF genes were found to be distributed in a head-to-tail fashion throughout the region with an average density of one ZNF duplicon every 150-180 kb of genomic distance. Sequence analysis of 208,967 bp of this region indicated the presence of two putative ZNF genes: one consisting of a novel member of this gene family (ZNF208) expressed ubiquitously in all tissues examined and the other representing a nonprocessed pseudogene (ZNF209), located 450 kb proximal to ZNF208. Large blocks of ( approximately 25-kb) inverted beta-satellite repeats with a remarkably symmetrical higher order repeat structure were found to bracket the functional ZNF gene. Hybridization analysis using the beta-satellite repeat as a probe indicates that beta-satellite interspersion between ZNF gene cassettes is a general property for 1.5 Mb of the ZNF gene cluster in 19p12. Both molecular clock data as well as a retroposon-mapping molecular fossil approach indicate that this ZNF cluster arose early during primate evolution (approximately 50 million years ago). We propose an evolutionary model in which heteromorphic pericentromeric repeat structures such as the beta satellites have been coopted to accommodate rapid expansion of a large gene family over a short period of evolutionary time. [The sequence data described in this paper have been submitted to GenBank under accession nos. AC003973 and AC004004.]