Nuclear receptors in nematodes: themes and variations.

Large-scale sequencing efforts are providing new perspectives on similarities and differences among species. Sequences encoding nuclear receptor (NR) transcription factors furnish one striking example of this. The three complete or nearly complete metazoan genome sequences - those of the nematode Caenorhabditis elegans, the fruit fly (Drosophila melanogaster) and the human - reveal dramatically different numbers of predicted NR genes: 270 for the nematode, 21 for the fruit fly and approximately 50 for the human. Although some classes of NRs present in insects and mammals are also represented among the nematode genes, most of the C. elegans NR sequences are distinct from those known in other phyla. Questions regarding the evolution and function of NR genes in nematodes, framed by t00e abundance and diversity of these genes in the C. elegans genome, are the focus of this article.

The nuclear receptor superfamily has undergone extensive proliferation and diversification in nematodes.

The nuclear receptor (NR) superfamily is the most abundant class of transcriptional regulators encoded in the Caenorhabditis elegans genome, with >200 predicted genes revealed by the screens and analysis of genomic sequence reported here. This is the largest number of NR genes yet described from a single species, although our analysis of available genomic sequence from the related nematode Caenorhabditis briggsae indicates that it also has a large number. Existing data demonstrate expression for 25% of the C. elegans NR sequences. Sequence conservation and statistical arguments suggest that the majority represent functional genes. An analysis of these genes based on the DNA-binding domain motif revealed that several NR classes conserved in both vertebrates and insects are also represented among the nematode genes, consistent with the existence of ancient NR classes shared among most, and perhaps all, metazoans. Most of the nematode NR sequences, however, are distinct from those currently known in other phyla, and reveal a previously unobserved diversity within the NR superfamily. In C. elegans, extensive proliferation and diversification of NR sequences have occurred on chromosome V, accounting for > 50% of the predicted NR genes.

Characterization of a muscle-associated antigen from Wuchereria bancrofti.

A recombinant clone, WbN1, isolated from a genomic expression library of Wuchereria bancrofti and showing restricted specificity at the DNA level (Southern and PCR analyses) for Wuchereria bancrofti and Brugia malayi has been previously described. Sequence analysis of WbN1 indicated that it had notable similarity to myosin. Further characterization using in situ hybridization has localized the mRNA in the muscle of the adult parasite and in the microfilariae. Rabbit polyclonal antiserum, raised against the recombinant WbN1 fused to the maltose-binding protein, recognized a 200-kDa polypeptide in immunoblots containing B. malayi antigen extracts. The same antibody also recognized myosin extracted from Brugia pahangi, Onchocerca volvulus, and Caenorhabditis elegans. Localization using the rabbit antiserum revealed the presence of the antigen in the adult muscle tissue and in the microfilariae; the same antibody inhibited the binding of a monoclonal antibody 28.2 (directed toward MHC B of C. elegans myosin) to the recombinant WbN1 antigen and also to purified C. elegans myosin. Based on homology data, structural location, competitive ELISA, and immunoblot we conclude that WbN1 is related to myosin or a similar myofibrillar protein.

Cloning of a cuticular antigen that contains multiple tandem repeats from the filarial parasite Dirofilaria immitis.

An unusual antigen composed of tandemly repeated protein units was cloned from the filarial parasite Dirofilaria immitis. The antigen was initially identified by screening a lambda gt11 cDNA library with serum from dogs immunized with irradiated D. immitis third-stage larvae. DNA sequence analysis of the cDNA clone, Di5, revealed a continuous open reading frame composed of two 399-base-pair repeats arranged in tandem. Southern blot analysis of genomic D. immitis DNA showed that the gene coding for Di5 is composed of a tandem array of 25-50 copies of this same 399-base-pair repeat. Antiserum raised against recombinant Di5 protein detected a protein "ladder," from about 14 to greater than 200 kDa with steps approximately 15 kDa apart, on immunoblots of D. immitis extract. Metabolic labeling of adult parasites with [35S]methionine showed that Di5 is synthesized as a large precursor that is subsequently cleaved to produce the ladder-like array. These results suggest that the characteristic ladder is created by proteolytic cleavage of the precursor at the same site in each monomer. The Di5 antigen was localized to the cuticle and hypodermis of adult D. immitis by immunoelectron microscopy. Both male and female parasites were found to release Di5 when cultured in vitro. DNA hybridization analysis demonstrated that Di5 is a member of a gene family present in many filarial parasites that infect both animal and human populations.

A recombinant clone of Wuchereria bancrofti with DNA specificity for human lymphatic filarial parasites.

In order to understand the immune response to Wuchereria bancrofti and to aid in the diagnosis of W. bancrofti infections, recombinant antigens were identified from a W. bancrofti genomic expression library made in lambda gt11 using a pool of sera from infected Indian patients. One of the recombinant clones, lambda WbN1, containing a 2.5-kb insert, reacted strongly to a pool of sera from patients with lymphatic filariasis but not to normal human sera. In addition, this clone showed restricted specificity at the genomic level to the major lymphatic filarial parasites W. bancrofti and Brugia malayi but not to the closely related filarial parasite Brugia pahangi or to other filarial and non-filarial species tested. Nucleotide sequence analysis indicated the cloned DNA to have homology to myosin-like myofibrillar proteins. Polymerase chain reaction amplification initiated by specific synthetic oligomers amplified DNA in a species-specific manner from as little as 16 pg of isolated DNA or from one microfilaria.

Nucleotide sequence of the phage lambda gt11 SacI-KpnI lacZ region.

The nucleotide sequence of the lambda gt11 SacI-KpnI region, surrounding the unique EcoRI cloning site, was directly determined. This sequence previously had to be compiled from several diverse sources. The direct sequence confirms the sequence predicted from the compilation and pinpoints other unique restriction enzyme targets in the region for use in subcloning.

An Escherichia coli vector to express and purify foreign proteins by fusion to and separation from maltose-binding protein.

A plasmid vector has been constructed that directs the synthesis of high levels (approximately 2% of total cellular protein) of fusions between a target protein and maltose-binding protein (MBP) in Escherichia coli. The MBP domain is used to purify the fusion protein in a one step procedure by affinity chromatography to crosslinked amylose resin. The fusion protein contains the recognition sequence (Ile-Glu-Gly-Arg) for blood coagulation factor Xa protease between the two domains. Cleavage by factor Xa separates the two domains and the target protein domain can then be purified away from the MBP domain by repeating the affinity chromatography step. A prokaryotic (beta-galactosidase) and a eukaryotic (paramyosin) protein have been successfully purified by this method.

Plasmid mapping computer program.

Three new computer algorithms are described which rapidly order the restriction fragments of a plasmid DNA which has been cleaved with two restriction endonucleases in single and double digestions. Two of the algorithms are contained within a single computer program (called MPCIRC). The Rule-Oriented algorithm, constructs all logical circular map solutions within sixty seconds (14 double-digestion fragments) when used in conjunction with the Permutation method. The program is written in Apple Pascal and runs on an Apple II Plus Microcomputer with 64K of memory. A third algorithm is described which rapidly maps double digests and uses the above two algorithms as adducts. Modifications of the algorithms for linear mapping are also presented.

Molecular weight determination program.

A computer program is described that will determine the molecular weight of DNA, RNA or protein molecules separated according to size by gel electrophoresis. It uses the sizes and migration distances of known molecules in a reference lane to compute a second or third order equation whose curve best fits the data points. It then computes the sizes of all molecules from this equation. Migration distances are measured and entered using an analog tablet. The program is written in Apple Pascal and designed to run on an Apple II Plus computer.

Stable chromosomal integration of the entire nitrogen fixation gene cluster from Klebsiella pneumoniae in yeast.

A bacterial plasmid containing the entire nitrogen fixation (nif) gene cluster (consisting of at least 15 genes) from Klebsiella pneumoniae was used in conjunction with an Escherichia coli-yeast shuttle plasmid containing the yeast his4 gene cluster to cotransform a his4- recipient strain of Saccharomyces cerevisiae. Of 87 histidine-independent clones screened, 2 contained nif DNA. Restriction and hybridization analyses showed that two copies of the nif plasmid (46 kilobases each) are integrated in tandem in the recipient chromosome by recombination between homologous regions in the transforming plasmids. Chromosomal integration was also verified by tetrad analysis, showing that the nif DNA behaved in meiosis like a Mendelian element. During mitotic growth, one of the two copies of the nif region is frequently lost. The remaining copy of nif is stable, even after 40 generations in nonselective medium.