The use of chromosomal translocations to study human immunoglobulin gene organization: mapping DH segments within 35 kb of the C mu gene and identification of a new DH locus.

We have studied the Burkitt's lymphoma cell line Daudi which carries the translocation t(8;14). The breakpoint of this translocation on the 14q+ chromosome occurs near to a rearranged DH-JH join, and the actual chromosome junction is a few hundred base pairs upstream of the joined DH element. The nucleotide sequence of the rearranged DH segment shows that it does not come from the previously described D cluster. Using this DH sequence as a probe we have identified two separate DH clusters. One of these is the major DH cluster and is located only 20 kb upstream of the JH segments. A pseudo-VH (probably the first VH segment) is also found approximately 98 kb from JH. A second, minor DH locus has been found which seems to be located on the distal side of the VH locus on chromosome 14, since there is little evidence for rearrangement or deletion of this locus in any B cell DNA analysed. A single VHIII subgroup gene is located within 25 kb of the newly identified DH element: it is possible that this minor locus occurs near the limit of the Igh locus.     

Nucleotide sequence of pvdD, a pyoverdine biosynthetic gene from Pseudomonas aeruginosa: PvdD has similarity to peptide synthetases.

Pseudomonas aeruginosa secretes a fluorescent siderophore, pyoverdine, when grown under iron-deficient conditions. Pyoverdine consists of a chromophoric group bound to a partly cyclic octapeptide. As a step toward understanding the molecular events involved in pyoverdine synthesis, we have sequenced a gene, pvdD, required for this process. The gene encodes a 2,448-residue protein, PvdD, which has a predicted molecular mass of 273,061 Da and contains two highly similar domains of about 1,000 amino acids each. The protein is similar to peptide synthetases from a range of bacterial and fungal species, indicating that synthesis of the peptide moiety of pyoverdine proceeds by a nonribosomal mechanism. The pvdD gene is adjacent to a gene, fpvA, which encodes an outer membrane receptor protein required for uptake of ferripyoverdine.     

Structural and transcriptional comparative analysis of the S locus regions in two self-incompatible Brassica napus lines

Self-incompatibility (SI) in Brassica is controlled by a single locus, termed the S locus. There is evidence that two of the S locus genes, SLG, which encodes a secreted glycoprotein, and SRK, which encodes a putative receptor kinase, are required for SI on the stigma side. The current model postulates that a pollen ligand recognizing the SLG/SRK receptors is encoded in the genomic region defined by the SLG and SRK genes. A fosmid contig of approximately 65 kb spanning the SLG-910 and SRK-910 genes was isolated from the Brassica napus W1 line. A new gene, SLL3, was identified using a novel approach combining cDNA subtraction and direct selection. This gene encodes a putative secreted small peptide and exists as multiple copies in the Brassica genome. Sequencing analysis of the 65-kb contig revealed seven additional genes and a transposon. None of these seven genes exhibited features expected of S genes on the pollen side. An approximately 88-kb contig of the A14 S region also was isolated from the B. napus T2 line and sequenced. Comparison of the two S regions revealed that (1) the gene organization downstream of SLG in both S haplotypes is highly colinear; (2) the distance between SLG-A14 and SRK-A14 genes is much larger than that between SLG-910 and SRK-910, with the intervening region filled with retroelements and haplotype-specific genes; and (3) the gene organization downstream of SRK in the two haplotypes is divergent. These observations lead us to propose that the SLG downstream region might be one border of the S locus and that the accumulation of heteromorphic sequences, such as retroelements as well as haplotype-unique genes, may act as a mechanism to suppress recombination between SLG and SRK.     

Comparative sequence analysis of VRN1 alleles of Lolium perenne with the co-linear regions in barley, wheat, and rice

Vernalization, a period of low temperature to induce transition from vegetative to reproductive state, is an important environmental stimulus for many cool season grasses. A key gene in the vernalization pathway in grasses is the VRN1 gene. The objective of this study was to identify causative polymorphism(s) at the VRN1 locus in perennial ryegrass (Lolium perenne) for variation in vernalization requirement. Two allelic Bacterial Artificial Chromosome clones of the VRN1 locus from the two genotypes Veyo and Falster with contrasting vernalization requirements were identified, sequenced, and characterized. Analysis of the allelic sequences identified an 8.6-kb deletion in the first intron of the VRN1 gene in the Veyo genotype which has low vernalization requirement. This deletion was in a divergent recurrent selection experiment confirmed to be associated with genotypes with low vernalization requirement. The region surrounding the VRN1 locus in perennial ryegrass showed microcolinearity to the corresponding region on chromosome 3 in Oryza sativa with conserved gene order and orientation, while the micro-colinearity to the corresponding region in Triticum monococcum was less conserved. Our study indicates that the first intron of the VRN1 gene, and in particular the identified 8.6?kb region, is an important regulatory region for vernalization response in perennial ryegrass.     

Characterization of a gene encoding an acetylase required for pyoverdine synthesis in Pseudomonas aeruginosa

Strains of Pseudomonas aeruginosa secrete one of three pyoverdine siderophores (types I to III). We have characterized a gene, pvdY(II) (for the pvdY gene present in type II P. aeruginosa strains), that is only present in strains that make type II pyoverdine. A mutation in pvdY(II) prevented pyoverdine synthesis. Bioinformatic, genetic, and biochemical approaches indicate that the PvdYII enzyme catalyzes acetylation of hydroxyornithine. Expression of pvdY(II) is repressed by the presence of iron and upregulated by the presence of type II pyoverdine. Characterization of pvdY(II) provides insights into the molecular basis for production of different pyoverdines by different strains of P. aeruginosa.     

Sea star populations diverge by positive selection at a sperm‐egg compatibility locus

Fertilization proteins of marine broadcast spawning species often show signals of positive selection. Among geographically isolated populations, positive selection within populations can lead to differences between them, and may result in reproductive isolation upon secondary contact. Here, we test for positive selection in the reproductive compatibility locus, bindin, in two populations of a sea star on either side of a phylogeographic break. We find evidence for positive selection at codon sites in both populations, which are under neutral or purifying selection in the reciprocal population. The signal of positive selection is stronger and more robust in the population where effective population size is larger and bindin diversity is greater. In addition, we find high variation in coding sequence length caused by large indels at two repetitive domains within the gene, with greater length diversity in the larger population. These findings provide evidence of population‐divergent positive selection in a fertilization compatibility locus, and suggest that sexual selection can lead to reproductive divergence between conspecific marine populations.     

Evidence of Gene Conversion in the Evolutionary Process of the Codon 41/42 (-CTTT) Mutation Causing β-Thalassemia in Southern China

The 4-bp deletion (-CTTT) at codon 41/42 (CD41/42) of the human beta-globin gene represents one of the most common beta-thalassemia mutations in East Asia and Southeast Asia, which is historically afflicted with endemic malaria, thus hypothetically evolving under natural selection by malaria infection. To understand the evolutionary process of generating the beta(CD41/42) allele and its maintenance, including the effect of natural selection on the pattern of linkage disequilibrium (LD), we sequenced a 15.933-kb region spanning 20.693 kb of the beta-globin cluster surrounding the 4-bp deletion using a sample from a Chinese population consisting of 24 normal individuals and 16 heterozygotes for the deletion. Forty-nine polymorphic sites were found. Analysis of the data, using a variety of methods including formal population genetics analysis and visual approaches, suggests that the spread of the CD41/42 (-CTTT) deletion is most likely mediated by interallelic gene conversion, although independent deletions in different lineages are also possible. The neutrality test resulted in a significant positive Tajima's D for the beta-globin locus, which is consistent with the existence of balancing selection. This suggests that the 4-bp deletion that occurred at this locus may be an event that is subject to natural selection, due to malaria, which leads to the heterozygote advantage, spread widely with further help by conversion and migration. The evolutionary process of this mutant through gene conversion that could conceivably take place between the 4-bp deletion and the normal sequence in the respective region is discussed in detail.     

Pyoverdine-mediated regulation of FpvA synthesis in Pseudomonas aeruginosa: involvement of a probable extracytoplasmic-function sigma factor,FpvI

A search of the pvd pyoverdine biosynthesis locus of Pseudomonas aeruginosa identified an open reading frame, PA2387, whose product exhibited a sequence similar to those of a number of so-called extracytoplasmic- function sigma factors responsible for siderophore-dependent expression of iron-siderophore receptors in Escherichia coli and Pseudomonas putida. Deletion of this gene, dubbed fpvI, compromised pyoverdine-dependent FpvA ferric pyoverdine receptor production and fpvA gene expression, while the cloned gene stimulated fpvA expression. A Fur-binding site was identified immediately upstream of fpvI, consistent with the observed iron-regulated expression of fpvI and fpvA.     

Adaptive evolution of UGT2B17 copy-number variation

The human UGT2B17 gene varies in copy number from zero to two per individual and also differs in mean number between populations from Africa, Europe, and East Asia. We show that such a high degree of geographical variation is unusual and investigate its evolutionary history. This required first reinterpreting the reference sequence in this region of the genome, which is misassembled from the two different alleles separated by an artifactual gap. A corrected assembly identifies the polymorphism as a 117 kb deletion arising by nonallelic homologous recombination between ~4.9 kb segmental duplications and allows the deletion breakpoint to be identified. We resequenced ~12 kb of DNA spanning the breakpoint in 91 humans from three HapMap and one extended HapMap populations and one chimpanzee. Diversity was unusually high and the time to the most recent common ancestor was estimated at ~2.4 or ~3.0 million years by two different methods, with evidence of balancing selection in Europe. In contrast, diversity was low in East Asia where a single haplotype predominated, suggesting positive selection for the deletion in this part of the world.     

FpvA receptor involvement in pyoverdine biosynthesis in Pseudomonas aeruginosa

Alignment of the Pseudomonas aeruginosa ferric pyoverdine receptor, FpvA, with similar ferric-siderophore receptors revealed that the mature protein carries an extension of ca. 70 amino acids at its N terminus, an extension shared by the ferric pseudobactin receptors of P. putida. Deletion of fpvA from the chromosome of P. aeruginosa reduced pyoverdine production in this organism, as a result of a decline in expression of genes (e.g., pvdD) associated with the biosynthesis of the pyoverdine peptide moiety. Wild-type fpvA restored pvd expression in the mutant, thereby complementing its pyoverdine deficiency, although a deletion derivative of fpvA encoding a receptor lacking the N terminus of the mature protein did not. The truncated receptor was, however, functional in pyoverdine-mediated iron uptake, as evidenced by its ability to promote pyoverdine-dependent growth in an iron-restricted medium. These data are consistent with the idea that the N-terminal extension plays a role in FpvA-mediated pyoverdine biosynthesis in P. aeruginosa.     

Detection of elastase production in Escherichia coli with the elastase structural gene from several non-elastase-producing strains of Pseudomonas aeruginosa.

The elastase structural gene from Pseudomonas aeruginosa IFO 3455 has been cloned and sequenced. Using this gene as a probe, we cloned the DNA fragments (pEL3080R, pEL10, and pEL103R) of the elastase gene from non-elastase-producing strains (P. aeruginosa IFO 3080, N-10, and PA103 respectively). These three Pseudomonas strains showed no detectable levels of elastase antigenicity by Western blotting (immunoblotting) or by elastase activity. When elastase structural genes about 8 kb in length were cloned into pUC18, an Escherichia coli expression vector, we were able to detect both elastase antigenicity and elastolytic activity in two bacterial clones (E. coli pEL10 and E. coli pEL103R). However, neither elastolytic activity nor elastase antigenicity was detected in the E. coli pEL3080R clone, although elastase mRNA was observed. The partial restriction map determined with several restriction enzymes of these three structural genes corresponded to that of P. aeruginosa IFO 3455. We sequenced the three DNA segments of the elastase gene from non-elastase-producing strains and compared the sequences with those from the elastase-producing P. aeruginosa strains IFO 3455 and PAO1. In P. aeruginosa N-10 and PA103, the sequences were almost identical to those from elastase-producing strains, except for several nucleotide differences. These minor differences may reflect a microheterogeneity of the elastase gene. These results suggest that two of the non-elastase-producing strains have the normal elastase structural gene and that elastase production is repressed by regulation of this gene expression in P. aeruginosa. Possible reasons for the lack of expression in these two strains are offered in this paper. In P. aeruginosa IFO 3080, the sequence had a 1-base deletion in the coding region, which should have caused a frameshift variation in the amino acid sequence. At present, we have no explanation for the abnormal posttransciptional behavior of this strain.     

Sequence analysis of the wall-associated protein precursor of Streptococcus mutans antigen A

The nucleotide sequence has been determined for the Streptococcus mutans wall-associated protein A (wapA) gene from serotype c strains Ingbritt and GS5. The nucleotide sequence for each wapA gene was virtually identical, although the gene from strain GS5 contained a 24 base pair deletion. A 29 amino acid signal peptide was specified by each wapA gene with a mature protein of 424 amino acids (Mr, 45,276) for strain Ingbritt and 416 amino acids (Mr, 44,846) for strain GS5. In the C-terminal region of the wall-associated protein A, considerable sequence similarity was found with the membrane anchor region of proteins from other Gram-positive organisms such as the group A streptococcal M protein and the group G streptococcal IgG binding protein. Adjacent to the proposed membrane anchor is a highly hydrophilic region which may span the cell wall; both sequence data and experimental evidence indicate the existence of a region immediately outside the wall at which proteolytic cleavage occurs to release antigen A of Mr 29,000 into the culture supernatant. Thus, the wall-associated protein A is a precursor of the 29,000 Mr antigen A.     

Diversity of the fsr-gelE region of the Enterococcus faecalis genome but conservation in strains with partial deletions of the fsr operon

Most Enterococcus faecalis isolates carry gelE, but many are gelatinase nonproducers due to the lack of fsrC (EF_1820) to EF_1841 (fsrC-EF_1841; 23.9 kb in strain V583), including most of the locus encoding Fsr, which activates gelE expression. Analysis of 22 accessible E. faecalis genomes revealed the identity of the 53-amino-acid propeptide of fsrD across multiple MLSTs (multilocus sequence types), although 12 distinctly different variations were found in the EF_1814-to-EF_1902 region. Diversity was seen in fsrABC, in the region EF_1814 to EF_1902, and in a 700-kb region surrounding fsrC-EF_1841. However, analysis of five sequenced strains carrying the fsrC-EF_1841 deletion and the putative integrative conjugative element efaB5 showed almost identical single nucleotide polymorphisms (SNPs) in gelE and an identical junction sequence, despite their unrelated MLSTs, in contrast to those shown by strains without the deletion. Further analysis confirmed the conserved gelE SNPs in 6 additional strains (11 in total) with the deletion. While we were unable to detect evidence of spontaneous deletion using OG1RF and 8 other strains, we were able to engineer a deletion of the 37-kb fsrC-EF_1841 region of OG1RF without deleterious effects, and the 37-kb mutant showed changes in biofilm and chaining similar to those shown by fsr-gelE mutants. In conclusion, we describe the identity of fsrD despite high plasticity within the fsrC-EF_1841 region and the surrounding sequence. However, strains lacking the fsrC-EF_1841 region show a distinct conservation of the sequence surrounding this deletion and in gelE, suggesting that the deletion may result from horizontal transfer and recombination.     

The 1.8 A crystal structure of PA2412, an MbtH-like protein from the pyoverdine cluster of Pseudomonas aeruginosa

Many bacteria use nonribosomal peptide synthetase (NRPS) proteins to produce peptide antibiotics and siderophores. The catalytic domains of the NRPS proteins are usually linked in large multidomain proteins. Often, additional proteins are coexpressed with NRPS proteins that modify the NRPS peptide products, ensure the availability of substrate building blocks, or play a role in the import or export of the NRPS product. Many NRPS clusters include a small protein of approximately 80 amino acids with homology to the MbtH protein of mycobactin synthesis in Mycobacteria tuberculosis; no function has been assigned to these proteins. Pseudomonas aeruginosa utilizes an NRPS cluster to synthesize the siderophore pyoverdine. The pyoverdine peptide contains a dihydroxyquinoline-based chromophore, as well as two formyl-N-hydroxyornithine residues, which are involved in iron binding. The pyoverdine cluster contains four modular NRPS enzymes and 10-15 additional proteins that are essential for pyoverdine production. Coexpressed with the pyoverdine synthetic enzymes is a 72-amino acid MbtH-like family member designated PA2412. We have determined the three-dimensional structure of the PA2412 protein and describe here the structure and the location of conserved regions. Additionally, we have further analyzed a deletion mutant of the PA2412 protein for growth and pyoverdine production. Our results demonstrate that PA2412 is necessary for the production or secretion of pyoverdine at normal levels. The PA2412 deletion strain is able to use exogenously produced pyoverdine, showing that there is no defect in the uptake or utilization of the iron-pyoverdine complex.     

Whole-genome sequence variation among multiple isolates of Pseudomonas aeruginosa

Whole-genome shotgun sequencing was used to study the sequence variation of three Pseudomonas aeruginosa isolates, two from clonal infections of cystic fibrosis patients and one from an aquatic environment, relative to the genomic sequence of reference strain PAO1. The majority of the PAO1 genome is represented in these strains; however, at least three prominent islands of PAO1-specific sequence are apparent. Conversely, approximately 10% of the sequencing reads derived from each isolate fail to align with the PAO1 backbone. While average sequence variation among all strains is roughly 0.5%, regions of pronounced differences were evident in whole-genome scans of nucleotide diversity. We analyzed two such divergent loci, the pyoverdine and O-antigen biosynthesis regions, by complete resequencing. A thorough analysis of isolates collected over time from one of the cystic fibrosis patients revealed independent mutations resulting in the loss of O-antigen synthesis alternating with a mucoid phenotype. Overall, we conclude that most of the PAO1 genome represents a core P. aeruginosa backbone sequence while the strains addressed in this study possess additional genetic material that accounts for at least 10% of their genomes. Approximately half of these additional sequences are novel.     

Comparative and genetic analysis of the porcine glucocerebrosidase (GBA) gene

The genomic sequence of the porcine (Sus scrofa) glucocerebrosidase (GBA) gene (5.7 kb), encoding glucocerebrosidase (glucosylceramidase; acid beta-glucosidase; EC 3.2.1.45), was determined and compared with human (Homo sapiens) GBA and GBAP (pseudogene). The porcine gene harbours 11 exons and 10 introns, and the genomic organization is identical with human GBA. The exon sequences, coding for signal peptide and mature protein, show 81% and 90% sequence identity, respectively, with the corresponding human GBA sequences. Short interspersed elements, SINEs (PREs), are present in introns 2, 4 and 7. There is no evidence of a pseudogene in pig. The deduced protein sequence of GBA consists of 39 amino acids of signal peptide (long form) and 497 amino acids of the mature protein; the latter shows 90% sequence identity with the human protein. Four polymorphisms were observed within the porcine gene: insertion/deletion of one of the two SINEs (PREs) in intron 2 (locus PREA); deletion of a 37- to 39-bp stretch in intron 4 (one direct repeat and 5′ end of PRE); deletion of a 47-bp stretch in the middle part of PRE in intron 4 (locus PREB); and single-base transition (C–T) in intron 6 (locus HaeIII–RFLP). GBA was assigned to chromosome 4q21 by FISH and was localized to the same region by linkage analysis and RH mapping, i.e., to the chromosome 4 segment where quantitative trait loci for growth and some carcass traits are located.     

Distribution and evolution of ferripyoverdine receptors in Pseudomonas aeruginosa

Pseudomonas aeruginosa is a ubiquitous Gram-negative bacterium, which is also able to cause severe opportunistic infections in humans. The colonization of the host is importantly affected by the production of the high-affinity iron (III) scavenging peptidic siderophore pyoverdine. The species P. aeruginosa can be divided into three subgroups ('siderovars'), each characterized by the production of a specific pyoverdine and receptor (FpvA). We used a multiplex PCR to determine the FpvA siderovar on 345 P. aeruginosa strains from environmental or clinical origin. We found about the same proportion of each type in clinical strains, while FpvA type I was slightly over-represented (49%) in environmental strains. Our multiplex PCR also detected the presence or absence of an additional receptor for type I pyoverdine (FpvB). The fpvB gene was in fact present in the vast majority of P. aeruginosa strains (93%), regardless of their siderovar or their origin. Finally, molecular analyses of fpvA and fpvB genes highlighted a complex evolutionary history, probably linked to the central role of iron acquisition in the ecology and virulence of P. aeruginosa .