Filarial and Wolbachia genomics.

Filarial nematode parasites, the causative agents for a spectrum of acute and chronic diseases including lymphatic filariasis and river blindness, threaten the well-being and livelihood of hundreds of millions of people in the developing regions of the world. The 2007 publication on a draft assembly of the 95-Mb genome of the human filarial parasite Brugia malayi- representing the first helminth parasite genome to be sequenced - has been followed in rapid succession by projects that have resulted in the genome sequencing of six additional filarial species, seven nonfilarial nematode parasites of animals and nearly 30 plant parasitic and free-living species. Parallel to the genomic sequencing, transcriptomic and proteomic projects have facilitated genome annotation, expanded our understanding of stage-associated gene expression and provided a first look at the role of epigenetic regulation of filarial genomes through microRNAs. The expansion in filarial genomics will also provide a significant enrichment in our knowledge of the diversity and variability in the genomes of the endosymbiotic bacterium Wolbachia leading to a better understanding of the genetic principles that govern filarial-Wolbachia mutualism. The goal here is to provide an overview of the trends and advances in filarial and Wolbachia genomics.

Determination of Wolbachia genome size by pulsed-field gel electrophoresis.

Genome sizes of six different Wolbachia strains from insect and nematode hosts have been determined by pulsed-field gel electrophoresis of purified DNA both before and after digestion with rare-cutting restriction endonucleases. Enzymes SmaI, ApaI, AscI, and FseI cleaved the studied Wolbachia strains at a small number of sites and were used for the determination of the genome sizes of wMelPop, wMel, and wMelCS (each 1.36 Mb), wRi (1.66 Mb), wBma (1.1 Mb), and wDim (0.95 Mb). The Wolbachia genomes studied were all much smaller than the genomes of free-living bacteria such as Escherichia coli (4.7 Mb), as is typical for obligate intracellular bacteria. There was considerable genome size variability among Wolbachia strains, especially between the more parasitic A group Wolbachia infections of insects and the mutualistic C and D group infections of nematodes. The studies described here found no evidence for extrachromosomal plasmid DNA in any of the strains examined. They also indicated that the Wolbachia genome is circular.

Denaturing polyacrylamide gel electrophoresis.

Polyacrylamide gels that contain a high concentration of urea as a denaturant are capable of resolving short (<500 nucleotides) single-stranded fragments of DNA or RNA that differ in length by as little as one nucleotide. Such gels are commonly used for DNA sequence analysis, as well as in PCR amplification of SSLPs (simple sequence length polymorphisms) for genotyping, genotyping by the ligase chain reaction (LCR), analysis of mutations by RNase A cleavage, chemical cleavage of heteroduplex DNA to identify mutations, and molecular analysis of fragile X syndrome by PCR. This appendix presents a protocol for the pouring, running, and processing of a typical gel which is 40-cm long with a uniform thickness of 0.4 mm, containing 7 M urea and 4% to 8% acrylamide.

Denaturing polyacrylamide gel electrophoresis.

Thin polyacrylamide gels that contain a high concentration of urea as a denaturant are capable of resolving short (<500 nucleotides) single-stranded fragments of DNA or RNA that differ in length by as little as one nucleotide. Such gels are uniquely suited for nucleic acid sequence analysis, which is required, for instance, for all footprinting protocols. Thicker gels are often used to purify oligonucleotides. This appendix describes the pouring, running, and processing of a typical sequencing gel, which is 40 cm long with a uniform thickness of 0.4 mm, containing 7 M urea and 4% to 8% acrylamide.

Preparation of templates for DNA sequencing.

This unit contains protocols for preparing DNA suitable for use as dideoxy sequencing templates and as material for end labeling and chemical sequencing. In all protocols, the starting material contains the recombinant molecule to be sequenced. DNA from M13mp-derived phage is easily prepared and is currently the most reliable source of template for large-scale dideoxy sequencing projects. Because it is occasionally necessary or convenient to use a lambda-derived phage as a source of DNA, a protocol for preparing lambda phage DNA from plate lysates is provided. Two protocols for minipreps of plasmid DNA are provided, one intended for dideoxy sequencing, the other for end labeling and chemical sequencing; they differ primarily in the way in which cellular RNA is removed. Alkali denaturation of double-stranded DNA (necessary prior to annealing) is described, and a final protocol describes the preparation of template for thermal cycle sequencing from a single phage plaque or bacterial colony.

DNA sequencing by the dideoxy method.

In the basic dideoxy sequencing reaction, an oligonucleotide primer is annealed to a single-stranded DNA template and extended by DNA polymerase in the presence of four deoxyribonucleoside triphosphates (dNTPs), one of which is 35S-labeled. The reaction also contains one of four dideoxyribonucleoside triphosphates (ddNTPs), which terminate elongation when incorporated into the growing DNA chain. After completion of the sequencing reactions, the products are subjected to electrophoresis on a high-resolution denaturing polyacrylamide gel and then autoradiographed to visualize the DNA sequence. Three variations of the dideoxy sequencing procedure are currently in use and are presented in this unit. In the "labeling/termination" procedure, primer chains are initially extended and labeled in the absence of terminating ddNTPs, whereas in the traditional "Sanger" procedure, labeling and termination of primer chains occur in a single step. A recent variation of the dideoxy sequencing method is thermal cycle sequencing in which the reaction mixture, containing template DNA, primer, thermostable DNA polymerase, dNTPs, and ddNTPs, is subjected to repeated rounds of denaturation, annealing, and elongation steps. The resulting linear amplification of the sequencing products allows much less template DNA to be used and eliminates independent primer annealing and template denaturation steps, which are required for the labeling/termination or Sanger procedures. The use of automated fluorescent sequencers for four-color dideoxy DNA sequencing is also described in detail.

Denaturing gel electrophoresis for sequencing.

The accuracy of DNA sequence determination depends largely upon resolution of the sequencing products in denaturing polyacrylamide gels. This unit provides a detailed description of the setup, electrophoresis, and processing of such gels. In general, the gels required for DNA sequencing are 40-cm long, of uniform thickness, and contain 4% to 8% acrylamide and 7 M urea. Modifications of this protocol increase the length of readable sequence information which can be obtained from a single gel (i.e., forming the gel with wedge-shaped spacers to create a field gradient, or incorporating a buffer gradient, an electrolyte gradient, or an acrylamide step gradient into the gel). A modification to the Basic Protocol--inclusion of formamide in the sequencing gel--is designed to overcome gel compressions arising from secondary structure in the sequencing products during gel electrophoresis. A discussion of acrylamide concentrations and electrophoresis conditions is included in the Commentary.

Alternative splicing creates sex-specific transcripts and truncated forms of the furin protease in the parasite Dirofilaria immitis.

Many extracellular proteins are activated by specific cleavage with an endoprotease. In nematodes, several proteins are cleaved after RX(K/R)R, the recognition site for the subtilisin-like proprotein convertases, furin and blisterase. To characterize furin in the parasitic nematode Dirofilaria immitis, we determined the sequence of the difur gene and its multiple transcripts. The gene spans 11 kb; encodes 16 exons and has a complex pattern of alternative splicing which generates at least 16 distinct mRNAs. The major transcript is a 4.4 kb mRNA which codes for a protein of 834 aa with an unusually long prodomain of 254 aa. Sex-specific splice variants of difur were observed by RT-PCR. The three female-specific and five male-specific transcripts are the first reported examples of sex-specific splicing in parasitic nematodes. This suggests that nematodes have sex-specific factors which regulate RNA splicing. Other splice variants are predicted to alter the phosphorylation and localization of the protease. Alternative splicing after the prodomain encodes a truncated protein that may be an inhibitor and/or substrate of Difurin.

Chemiluminescent detection of sequential DNA hybridizations to high-density, filter-arrayed cDNA libraries: a subtraction method for novel gene discovery.

A chemiluminescent approach for sequential DNA hybridizations to high-density filter arrays of cDNAs, using a biotin-based random priming method followed by a streptavidin/alkaline phosphatase/CDP-Star detection protocol, is presented. The method has been applied to the Brugia malayi genome project, wherein cDNA libraries, cosmid and bacterial artificial chromosome (BAC) libraries have been gridded at high density onto nylon filters for subsequent analysis by hybridization. Individual probes and pools of rRNA probes, ribosomal protein probes and expressed sequence tag probes show correct specificity and high signal-to-noise ratios even after ten rounds of hybridization, detection, stripping of the probes from the membranes and rehybridization with additional probe sets. This approach provides a subtraction method that leads to a reduction in redundant DNA sequencing, thus increasing the rate of novel gene discovery. The method is also applicable for detecting target sequences, which are present in one or only a few copies per cell; it has proven useful for physical mapping of BAC and cosmid high-density filter arrays, wherein multiple probes have been hybridized at one time (multiplexed) and subsequently "deplexed" into individual components for specific probe localizations.

Cloning and characterization of the Bg/II restriction-modification system reveals a possible evolutionary footprint.

Bg/II, a type II restriction-modification (R-M) system from Bacillus globigii, recognizes the sequence 5'-AGATCT-3'. The system has been cloned into E. coli in multiple steps: first the methyltransferase (MTase) gene, bglIIM, was cloned from B. globigii RUB561, a variant containing an inactivated endonuclease (ENase) gene (bglIIR). Next the ENase protein (R.BglII) was purified to homogeneity from RUB562, a strain expressing the complete R-M system. Oligonucleotide probes specific for the 5' end of the gene were then synthesized and used to locate bglIIR, and the gene was isolated and cloned in a subsequent step. The nucleotide sequence of the system has been determined, and several interesting features have been found. The genes are tandemly arranged, with bglIIR preceding bglIIM. The amino acid sequence of M.BglII is compared to those of other known MTases. A third gene encoding a protein with sequence similarity to known C elements of other R-M systems is found upstream of bglIIR. This is the first instance of a C gene being associated with an R-M system where the R and M genes are collinear. In addition, open reading frames (ORFs) resembling genes involved with DNA mobility are found in close association with BglII. These may shed light on the evolution of the R-M system.

Thermal cycle dideoxy DNA sequencing.

Thermal cycle dideoxy DNA sequencing eliminates the requirements for independent primer annealing and double-stranded DNA denaturation steps. The method enables sequencing from nanogram amounts of DNA from double-stranded and single-stranded PCR products, and plasmid or phage DNA templates. Thermal cycle sequencing also enables direct sequencing from bacterial colonies or phage plaques. Protocols using the Vent exo- DNA polymerase, helpful suggestions, and a troubleshooting guide are also presented.

Cloning, expression and sequence analysis of the SphI restriction-modification system.

SphI, a type II restriction-modification (R-M) system from the bacterium Streptomyces phaeochromogenes, recognizes the sequence 5'-GCATGC. The SphI methyltransferase (MTase)-encoding gene, sphIM, was cloned into Escherichia coli using MTase selection to isolate the clone. However, none of these clones contained the restriction endonuclease (ENase) gene. Repeated attempts to clone the complete ENase gene along with sphIM in one step failed, presumably due to expression of SphI ENase gene, sphIR, in the presence of inadequate expression of sphIM. The complete sphIR was finally cloned using a two-step process. PCR was used to isolate the 3' end of sphIR from a library. The intact sphIR, reconstructed under control of an inducible promoter, was introduced into an E. coli strain containing a plasmid with the NlaIII MTase-encoding gene (nlaIIIM). The nucleotide sequence of the SphI system was determined, analyzed and compared to previously sequenced R-M systems. The sequence was also examined for features which would help explain why sphIR unlike other actinomycete ENase genes seemed to be expressed in E. coli.

Cloning and sequence comparison of AvaI and BsoBI restriction-modification systems.

AvaI and BsoBI restriction endonucleases are isoschizomers which recognize the symmetric sequence 5'CYCGRG3' and cleave between the first C and second Y to generate a four-base 5' extension. The AvaI restriction endonuclease gene (avaIR) and methylase gene (avaIM) were cloned into Escherichia coli by the methylase selection method. The BsoBI restriction endonuclease gene (bsoBIR) and part of the BsoBI methylase gene (bsoBIM) were cloned by the "endo-blue" method (SOS induction assay), and the remainder of bsoBIM was cloned by inverse PCR. The nucleotide sequences of the two restriction-modification (RM) systems were determined. Comparisons of the predicted amino acid sequences indicated that AvaI and BsoBI endonucleases share 55% identity, whereas the two methylases share 41% identity. Although the two systems show similarity in protein sequence, their gene organization differs. The avaIM gene precedes avaIR in the AvaI RM system, while the bsoBI R gene is located upstream of bsoBI M in the BsoBI RM system. Both AvaI and BsoBI methylases contain motifs conserved among the N4 cytosine methylases.

The XmnI restriction-modification system: cloning, expression, sequence organization and similarity between the R and M genes.

The xmnIRM genes from Xanthomonas manihotis 7AS1 have been cloned and expressed in Escherichia coli. The nucleotide (nt) sequences of both genes were determined. The XmnI methyltransferase (MTase)-encoding gene is 1861 bp in length and codes for 620 amino acids (aa) (68660 Da). The restriction endonuclease (ENase)-encoding gene is 959 bp long and therefore codes for a 319-aa protein (35275 Da). The two genes are aligned tail to tail and they overlap at their respective stop codons About 4 x 10(4) units/g wet cell paste of R.XmnI was obtained following IPTG induction in a suitable E. coli host. The xmnIR gene is expressed from the T7 promoter. M.XmnI probably modifies the first A in the sequence, GAA(N)4TTC. The xmnIR and M genes contain regions of conserved similarity and probably evolved from a common ancestor. M.XmnI is loosely related to M.EcoRI. The XmnI R-M system and the type-I R-M systems probably derived from a common ancestor.

Cloning and expression of the NaeI restriction endonuclease-encoding gene and sequence analysis of the NaeI restriction-modification system.

NaeI, a type-II restriction-modification (R-M) system from the bacterium Nocardia aerocolonigenes, recognizes the sequence 5'-GCCGGC. The NaeI DNA methyltransferase (MTase)-encoding gene, naeIM, had been cloned previously in Escherichia coli [Van Cott and Wilson, Gene 74 (1988) 55-59]. However, none of these clones expressed detectable levels of the restriction endonuclease (ENase). The absence of the intact ENase-encoding gene (naeIR) within the isolated MTase clones was confirmed by recloning the MTase clones into Streptomyces lividans. The complete NaeI system was finally cloned using E. coli AP1-200 [Piekarowicz et al., Nucleic Acids Res. 19 (1991) 1831-1835] and less stringent MTase-selection conditions. The naeIR gene was expressed first by cloning into S. lividans, and later by cloning under control of a regulated promoter in an E. coli strain preprotected by the heterologous MspI MTase (M.MspI). The DNA sequence of the NaeI R-M system has been determined, analyzed and compared to previously sequenced R-M systems.

Organization and sequence of the SalI restriction-modification system.

The organization and nucleotide (nt) sequences were determined for the genes encoding the SalI restriction and modification (R-M) system (recognition sequence 5'-GTCGAC-3') from Streptomyces albus G. The system comprises two genes, salIR, coding for the restriction endonuclease (ENase, R.SalI; probably 315 amino acids (aa), a predicted M(r) of 35,305; product, G'TCGAC) and salIM, coding for the methyltransferase (MTase, M.SalI; probably 587 aa, a predicted M(r) of 64,943; product, GTCGm6AC). The genes are adjacent, they have the same orientation, and they occur in the order salIR then salIM. R.SalI contains a putative magnesium-binding motif similar to those at the active sites of R.EcoRI and R.EcoRV, but otherwise it bears little aa sequence similarity to other ENases. M.SalI is a member of the m6A gamma class of MTases. In aa sequence it resembles M.AccI, another m6A gamma-MTase whose recognition sequence includes the SalI recognition sequence as a subset.

Cloning, analysis and expression of the HindIII R-M-encoding genes.

The genes encoding the HindIII restriction endonuclease (R.HindIII ENase) and methyltransferase (M.HindIII MTase) from Haemophilus influenzae Rd were cloned and expressed in Escherichia coli and their nucleotide (nt) sequences were determined. The genes are transcribed in the same orientation, with the ENase-encoding gene (hindIIIR) preceding the MTase-encoding gene (hindIIIM). The two genes overlap by several nt. The ENase is predicted to be 300 amino acids (aa) in length (34,950 Da); the MTase is predicted to be 309 aa (35,550 Da). The HindIII ENase and MTase activities increased approx. 20-fold when the genes were brought under the control of an inducible lambda pL promoter. Highly purified HindIII ENase and MTase proteins were prepared and their N-terminal aa sequences determined. In H. influenzae Rd, the HindIII R-M genes are located between the holC and valS genes; they are not closely linked to the HindII R-M genes.