Isolation of HLA-DR1.(staphylococcal enterotoxin A)2 trimers in solution.

Mutational studies indicate that the superantigen staphylococcal enterotoxin A (SEA) has two separate binding sites for major histocompatibility complex (MHC) class II molecules. Direct evidence is provided here for the formation of SEA-MHC class II trimers in solution. Isoelectric focusing separated SEA-HLA-DR1 complexes into both dimers and HLA-DR1.SEA2 trimers. The molar ratio of components was determined by dual isotope labeling. The SEA mutant SEA-F47S, L48S, Y92A, which is deficient in MHC class II alpha-chain binding, formed only dimers with HLA-DR1, whereas a second SEA mutant, SEA-H225A, which lacks high-affinity MHC class II beta-chain binding was incapable of forming any complexes. Thus SEA binding to its MHC receptor is a two-step process involving initial beta-chain binding followed by cooperative binding of a second SEA molecule to the class II alpha chain.

Isolation and identification of a diuretic hormone from the mealworm Tenebrio molitor.

A diuretic hormone of unusual structure was isolated from extracts of whole heads of the mealworm Tenebrio molitor. The hormone is a 37-aa peptide of 4371 Da, with the sequence SPTISITAPIDVLRKTWEQERARKQMVKNREFLNSLN. This peptide increases cAMP production in Malpighian tubules of T. molitor. The amino acid sequence reveals that this peptide is a member of the family of sauvagine/corticotropin-releasing factor/urotensin I-related insect diuretic hormones. The C-terminal sequence of this peptide is quite different from other members of this family, which have a hydrophobic C terminus (isoleucinamide or valinamide). When aligned comparably, T. molitor diuretic hormone has a more hydrophilic C terminus, leucylasparagine (free acid). In contrast to all other known diuretic hormones of this family, this peptide has exceptionally low stimulatory activity on cAMP production in Malpighian tubules of Manduca sexta. However, at nanomolar concentrations it stimulates cAMP production in Malpighian tubules of T. molitor. Diuretic hormones of this family have been isolated previously from Lepidoptera, Orthoptera, Dictyoptera, and Diptera. This appears to be the first diuretic hormone isolated from a coleopteran insect.

Isolation and characterization of a pseudoautosomal region-specific genetic marker in C57BL/6 mice using genomic representational difference analysis.

Representational difference analysis was used to identify strain-specific differences in the pseudoautosomal region (PAR) of mouse X and Y chromosomes. One second generation (C57BL/6 x Mus spretus) x Mus spretus interspecific backcross male carrying the C57BL/6 (B6) PAR was used for tester DNA. DNA from five backcross males from the same generation that were M. spretus-type for the PAR was pooled for the driver. A cloned probe designated B6-38 was recovered that is B6-specific in Southern analysis. Analysis of genomic DNA from several inbred strains of laboratory mice and diverse Mus species and subspecies identified a characteristic Pst I pattern of fragment sizes that is present only in the C57BL family of strains. Hybridization was observed with sequences in DBA/2J and to a limited extent with Mus musculus (PWK strain) and Mus castaneus DNA. No hybridization was observed in DNA of different Mus species, M. spretus, M. hortulanus, and M. caroli. Genetic analyses of B6-38 was conducted using C57BL congenic males that carry M. spretus alleles for distal X chromosome loci and the PAR and outcrosses of heterozygous congenic females with M. spretus. These analyses demonstrated that the B6-38 sequences were inherited with both the X and Y chromosome. B6-38 sequences were genetically mapped as a locus within the PAR using two interspecific backcrosses. The locus defined by B6-38 is designated DXYRp1. Preliminary analyses of recombination between the distal X chromosome gene amelogenin (Amg) and the PAR loci for either TelXY or sex chromosome association (Sxa) suggest that the locus DXYRp1 maps to the distal portion of the PAR.

Isolation of virus-neutralizing RNAs from a large pool of random sequences.

RNA and ribonuclease-resistant RNA analogs that bound and neutralized Rous sarcoma virus (RSV) were isolated from a large pool of random sequences by multiple cycles of in vitro selection using infectious viral particles. The selected RNA pool of RSV-binding sequences at a concentration of 0.16 microM completely neutralized the virus. Of 19 sequences cloned from the selected pool, 5 inhibited RSV infection. The selected RNA and RNA analogs were shown to neutralize RSV by interacting with the virus, rather than by adversely affecting the host cells. The selection of the anti-RSV RNA and RNA analogs by intact virions immediately suggests the potential application of this approach to develop RNA and RNA analogs as inhibitors of other viruses such as human immunodeficiency virus.

Isolation of yeast artificial chromosomes free of endogenous yeast chromosomes: construction of alternate hosts with defined karyotypic alterations.

An intrinsic feature of yeast artificial chromosomes (YACs) is that the cloned DNA is generally in the same size range (i.e., approximately 200-2000 kb) as the endogenous yeast chromosomes. As a result, the isolation of YAC DNA, which typically involves separation by pulsed-field gel electrophoresis, is frequently confounded by the presence of a comigrating or closely migrating endogenous yeast chromosome(s). We have developed a strategy that reliably allows the isolation of any YAC free of endogenous yeast chromosomes. Using recombination-mediated chromosome fragmentation, a set of Saccharomyces cerevisiae host strains was systematically constructed. Each strain contains defined alterations in its electrophoretic karyotype, which provide a large-size interval devoid of endogenous chromosomes (i.e., a karyotypic "window"). All of the constructed strains contain the kar1-delta 15 mutation, thereby allowing the efficient transfer of a YAC from its original host into an appropriately selected window strain using the kar1-transfer procedure. This approach provides a robust and efficient means to obtain relatively pure YAC DNA regardless of YAC size.

Isolation, regulation, and DNA-binding properties of three Drosophila nuclear hormone receptor superfamily members.

We have designed a rapid cloning and screening strategy to identify new members of the nuclear hormone receptor superfamily that are expressed during the onset of Drosophila metamorphosis. Using this approach, we isolated three Drosophila genes, designated DHR38, DHR78, and DHR96. All three genes are expressed throughout third-instar larval and prepupal development. DHR38 is the Drosophila homolog of NGFI-B and binds specifically to an NGFI-B response element. DHR78 and DHR96 are orphan receptor genes. DHR78 is induced by 20-hydroxyecdysone (20E) in cultured larval organs, and its encoded protein binds to two AGGTCA half-sites arranged as either direct or palindromic repeats. DHR96 is also 20E-inducible, and its encoded protein binds selectively to the hsp27 20E response element. The 20E receptor can bind to each of the sequences recognized by DHR78 and DHR96, indicating that these proteins may compete with the receptor for binding to a common set of target sequences.

Localization of pheromonal sexual dimorphism in Drosophila melanogaster and its effect on sexual isolation.

Drosophila melanogaster is sexually dimorphic for cuticular hydrocarbons, with males and females having strikingly different profiles of the long-chain compounds that act as contact pheromones. Gas-chromatographic analysis of sexual mosaics reveals that the sex specificity of hydrocarbons is located in the abdomen. This explains previous observations that D. melanogaster males display the strongest courtship toward mosaics with female abdomens. We also show that males of the sibling species Drosophila simulans preferentially court D. melanogaster mosaics with male abdomens. Because the primary male hydrocarbon in D. melanogaster is also the primary female hydrocarbon in D. simulans, this supports the idea that interspecific differences in cuticular hydrocarbons contribute to sexual isolation.

Isolation of cDNA clones encoding RICH: a protein induced during goldfish optic nerve regeneration with homology to mammalian 2',3'-cyclic-nucleotide 3'-phosphodiesterases.

Using data derived from peptide sequencing of p68/70, a protein doublet induced during optic nerve regeneration in goldfish, we have isolated cDNAs that encode RICH (regeneration-induced CNPase homolog) from a goldfish regenerating retina cDNA library. The predicted RICH protein comprises 411 amino acids, possesses a pI of 4.48, and shows significant homology to the mammalian myelin marker enzyme 2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNPase; EC 3.1.4.37). The mRNA encoding RICH was demonstrated, by both Northern blot analysis and RNase protection assays, to be induced as much as 8-fold in regenerating goldfish retinas at 20 days after nerve crush. Analysis of total RNA samples from various tissues showed a broad distribution of RICH mRNA, with the highest levels observed in gravid ovary. The data obtained strongly suggest that RICH is identical or very similar to p68/70. The molecular cloning of RICH provides the means for a more detailed analysis of its function in nerve regeneration. Additionally, the homology of RICH and CNPase suggests that further investigation may provide additional insight into the role of these proteins in the nervous system.

Isolation and characterization of pokeweed antiviral protein mutations in Saccharomyces cerevisiae: identification of residues important for toxicity.

Pokeweed antiviral protein (PAP), a 29-kDa protein isolated from Phytolacca americana inhibits translation by catalytically removing a specific adenine residue from the 28S rRNA of eukaryotic ribosomes. PAP has potent antiviral activity against many plant and animal viruses, including human immunodeficiency virus. We describe here development of a positive selection system to isolate PAP mutants with reduced toxicity. In vitro translation in the presence or absence of microsomal membranes shows that PAP is synthesized as a precursor and undergoes at least two different proteolytic processing steps to generate mature PAP. The PAP cDNA was placed under control of the galactose-inducible GAL1 promoter and transformed into Saccharomyces cerevisiae. Induction of PAP expression was lethal to yeast. The PAP expression plasmid was mutagenized and plasmids encoding mutant PAP genes were identified by their failure to kill S. cerevisiae. A number of mutant alleles were sequenced. In one mutant, a point mutation at Glu-177 inactivated enzymatic function in vitro, suggesting that this glutamic acid residue is located at or near the catalytic site. Mutants with either point mutations near the N terminus or a nonsense mutation at residue 237 produced protein that was enzymatically active in vitro, suggesting that the toxicity of PAP is not due solely to enzymatic activity. Toxicity of PAP appears to be a multistep process that involves possibly different domains of the protein.

Isolation of a primate embryonic stem cell line.

Embryonic stem cells have the ability to remain undifferentiated and proliferate indefinitely in vitro while maintaining the potential to differentiate into derivatives of all three embryonic germ layers. Here we report the derivation of a cloned cell line (R278.5) from a rhesus monkey blastocyst that remains undifferentiated in continuous passage for > 1 year, maintains a normal XY karyotype, and expresses the cell surface markers (alkaline phosphatase, stage-specific embryonic antigen 3, stage-specific embryonic antigen 4, TRA-1-60, and TRA-1-81) that are characteristic of human embryonal carcinoma cells. R278.5 cells remain undifferentiated when grown on mouse embryonic fibroblast feeder layers but differentiate or die in the absence of fibroblasts, despite the presence of recombinant human leukemia inhibitory factor. R278.5 cells allowed to differentiate in vitro secrete bioactive chorionic gonadotropin into the medium, express chorionic gonadotropin alpha- and beta-subunit mRNAs, and express alpha-fetoprotein mRNA, indicating trophoblast and endoderm differentiation. When injected into severe combined immunodeficient mice, R278.5 cells consistently differentiate into derivatives of all three embryonic germ layers. These results define R278.5 cells as an embryonic stem cell line, to our knowledge, the first to be derived from any primate species.

Photocleavable biotin derivatives: a versatile approach for the isolation of biomolecules.

While the strong biotin-avidin interaction has been widely used for the detection of biomolecules, its irreversibility complicates their isolation. We report the synthesis of a photocleavable biotin derivative (PCB) which eliminates many limitations of existing methods. This reagent contains a biotin moiety linked through a spacer arm to a photocleavable moiety, which reacts selectively with primary amino groups on any substrate. In experiments using [leucine]-enkephalin as a model substrate, we show that PCB retains its high affinity toward avidin/streptavidin and allows rapid (< 5 min) and efficient (> 99%) photorelease of the substrate in a completely unaltered form. Photocleavable biotins should be useful in numerous applications involving the isolation of proteins, nucleic acids, lipids, and cells.

Isolation of multiple sequences from the Plasmodium falciparum genome that encode conserved domains homologous to those in erythrocyte-binding proteins.

Open reading frames in the Plasmodium falciparum genome encode domains homologous to the adhesive domains of the P. falciparum EBA-175 erythrocyte-binding protein (eba-175 gene product) and those of the Plasmodium vivax and Plasmodium knowlesi Duffy antigen-binding proteins. These domains are referred to as Duffy binding-like (DBL), after the receptor that determines P. vivax invasion of Duffy blood group-positive human erythrocytes. Using oligonucleotide primers derived from short regions of conserved sequence, we have developed a reverse transcription-PCR method that amplifies sequences encoding the DBL domains of expressed genes. Products of these reverse transcription-PCR amplifications include sequences of single-copy genes (including eba-175) and variably transcribed genes that cross-hybridize to multiple regions of the genome. Restriction patterns of the multicopy genes show a high degree of polymorphism among different parasite lines, whereas single-copy genes are generally conserved. Characterization of the single-copy genes has identified a gene (ebl-1) that is related to eba-175 and is likely to be involved in erythrocyte invasion.

Isolation of small polarized bile duct units.

Fragments of small interlobular bile ducts averaging 20 microns in diameter can be isolated from rat liver. These isolated bile duct units form luminal spaces that are impermeant to dextran-40 and expand in size when cultured in 10 microM forskolin for 24-48 hr. Secretion is Cl- and HCO3- dependent and is stimulated by forskolin > dibutyryl cAMP > secretion but not by dideoxyforskolin, as assessed by video imaging techniques. Secretin stimulates Cl-/HCO3- exchange activity, and intraluminal pH increases after forskolin administration. These studies establish that small polarized physiologically intact interlobular bile ducts can be isolated from rat liver. These isolated bile duct units should be useful preparations for assessing the transport properties of small bile duct segments, which are the primary site of injury in cholestatic liver disorders, known as "vanishing bile duct syndromes."

A chimeric light-regulated amino acid transport system allows the isolation of blue light regulator (blr) mutants of Neurospora crassa.

We have developed a system for the isolation of Neurospora crassa mutants that shows altered responses to blue light. To this end we have used the light-regulated promoter of the albino-3 gene fused to the neutral amino acid permease gene mtr. The product of the mtr gene is required for the uptake of neutral aliphatic and aromatic amino acids, as well as toxic analogs such as p-flurophenylalanine or 4-methyltryptophan. mtr trp-2-carrying cells were transformed with the al-3 promoter-mtr wild-type gene (al-3p-mtr+) to obtain a strain with a light-regulated tryptophan uptake. This strain is sensitive to p-fluorophenylalanine when grown under illumination and resistant when grown in the dark. UV mutagenesis of the al-3p-mtr(+)-carrying strain allowed us to isolate two mutant strains, BLR-1 and BLR-2 (blue light regulator), that are light-resistant to p-fluorophenylalanine and have lost the ability to grow on tryptophan. These two strains have a pale-orange phenotype and show down-regulation of all the photoregulated genes tested (al-3, al-1, con-8, and con-10). Mutations in the BLR strains are not allelic with white collar 1 or white collar 2, regulatory genes that are also involved in the response to blue light.

Direct detection and isolation of restriction landmark genomic scanning (RLGS) spot DNA markers tightly linked to a specific trait by using the RLGS spot-bombing method.

We have developed a technique for isolating DNA markers tightly linked to a target region that is based on RLGS, named RLGS spot-bombing (RLGS-SB). RLGS-SB allows us to scan the genome of higher organisms quickly and efficiently to identify loci that are linked to either a target region or gene of interest. The method was initially tested by analyzing a C57BL/6-GusS mouse congenic strain. We identified 33 variant markers out of 10,565 total loci in a 4.2-centimorgan (cM) interval surrounding the Gus locus in 4 days of laboratory work. The validity of RLGS-SB to find DNA markers linked to a target locus was also tested on pooled DNA from segregating backcross progeny by analyzing the spot intensity of already mapped RLGS loci. Finally, we used RLGS-SB to identify DNA markers closely linked to the mouse reeler (rl) locus on chromosome 5 by phenotypic pooling. A total of 31 RLGS loci were identified and mapped to the target region after screening 8856 loci. These 31 loci were mapped within 11.7 cM surrounding rl. The average density of RLGS loci located in the rl region was 0.38 cM. Three loci were closely linked to rl showing a recombination frequency of 0/340, which is < 1 cM from rl. Thus, RLGS-SB provides an efficient and rapid method for the detection and isolation of polymorphic DNA markers linked to a trait or gene of interest.