Preparation and Properties of Nanoparticles, tRNA-Bivalent Metal Cation (Me2+) Complexes, and Prospects of Their Practical Use.

The patterns of formation of RNA nanoparticles (NPs) during thermal cycling of bacterial total tRNA in the presence of cations Ca2+, Mn2+, Ni2+, Zn2+, Co2+, and Cu2+ were studied. The optimal conditions for the production of NPs were found, and it was revealed that their size depends on the ratio of the concentrations of Me2+ and tRNA. The concentration of reagents for obtaining NPs of small size (from 5 to 100 nm) was selected. It was shown that tRNA-based nanoparticles can comprise short (20-50 nt) ribooligonucleotides, including aptamers and siRNAs. The stability of NPs during storage in buffer solutions of various composition was studied. It was found that the initial suspensions of NPs are quite stable, but they are rapidly destroyed in PBS buffer (pH 7.4). A simple and effective stabilizer (polyarginine) was found, the additives of which ensure the preservation of nanoparticles in PBS buffer for more than 5 h. Nanoparticles modified with the stabilizer are resistant to blood serum nucleases and can be used for transfection.

The structure-forming role of magnesium pyrophosphate in the formation of DNA-containing microparticles during PCR.

This work is devoted to studying the mechanisms of formation of DNA-containing microparticles (MPs) during PCR. It was found that pyrophosphate, a byproduct of DNA synthesis, and magnesium cations are required for their formation, as evidenced by the results of biochemical and electron microscopy studies.

[Micro- and nanoparticles of condensed DNA formed in a PCR with yeast genomic DNA as a template. Electron microscopy data].

It has been previously found that in a PCR with yeast genomic DNA as a template, microparticles of condensed DNA are formed in the presence of KlenTaq polymerase. In the present work, the study of these microparticles was continued using electron microscopy. It was shown that along with standard electron-dense microspheres, microspheres of a low electron density with a few thorns or without any thorns are formed. Various types of nanoparticles were detected in the samples: nanowires, dot-like electron-absorbing particles (nanodots), and compact nanoparticles (nanoscales) of different shape and size. It was found that increasing the number of PCR cycles above the optimum leads to an abrupt rise in the amount of nanoparticles in the PCR mixture. Suspensions of microparticles after quick (5 min) heating at 94 degrees C were examined. The partial melting of the microspheres in the heated samples was established: they lost part of the DNA and decreased in size; simultaneously, abundant clusters of nanowires appeared. The effect of nuclease S1 on the DNA of microspheres was studied. The molecular mechanisms of the formation of micro- and nanoparticles are discussed.

[Condensed DNA particles formed in PCR with plasmid DNA: electron microscopy study].

An electron microscopy study of large-sized DNA microparticles produced in PCR with different gene-specific primers and plasmid DNAs is described. DNA microspheres of two distinct types were revealed in the all studied samples, namely smooth moderately electron-dense microspheres, and highly electron-dense particles with large thorns and offshoots. Singular microspheres have the average diameter of 1 mum, and their aggregates were up to 3 mum in dimensions. In addition, rare so-called three-dimensional net-like structures with various size (up to several micrometers) were observed. They consisted of different amounts of DNA nanoparticles, having the special compact topology. In some studied samples the discs (nanodiscs) of several dozens nm in thickness and up to 3 mum in diameter were revealed. It was shown that the quantity of net-like structures and nanodiscs sharply increases in asymmetric PCR. We also observed DNA nanowires of different length and thickness, nanodots, nanoparticles in the form of shits of paper as well as electron-dense spherical nanoparticles of big size. Aqueous suspensions of DNA microparticles were heated at 94 degrees C for 5 min and analyzed by electron microscopy. It was shown that microspheres in heated suspensions underwent partial melting; they lost a part of DNA, therefore details of their structure (ultrastructure) can be recognized. At the some time numerous tangles of nanowires appeared. Molecular mechanisms of the DNA micro- and nanoparticles formation are discussed.

[Microparticles from coupled DNA formed in the process of polymerase chain reaction].

DNA microparticle formation in the course of a polymerase chain reaction (PCR) is reported. PCR with gene-specific and partially complementary primers and yeast genomic DNA as a template was shown to yield spherical DNA-composed microparticles as well as their aggregates and conglomerates, along with routine linear DNA. Microparticles were formed at late PCR stages and could be easily identified by the reaction with fluorescently labeled oligonucleotide primers or by staining of the PCR mixture with fluorescent dyes (acridine orange, propidium iodide or DAPI). According to the data of epifluorescent and electron microscopy, the microparticle size varied from 500 nm to 3-4 microm and the particles were multimeric star-shaped spheres or aggregates formed by several fused microspheres. Some properties of the microspheres were studied. It was found that the Mg2+ cations comprising the PCR buffer played a key role in the formation of microparticles and the stabilization of their structures.

[Rapid and efficient extraction of soluble proteins from gram-negative microorganisms without disruption of cell walls].

The ability of buffer solutions containing low concentrations of nonionic detergents (Triton X-100, Tween 20, Brij 58, and Lubrol PX) and the anionic detergent sodium deoxycholate, as well as mixtures of these detergents with chaeotropes (urea and guanidine hydrochloride), to extract intracellular proteins of Gram-negative microorganisms (Escherichia coli and Pseudomonas aeruginosa) was studied. It was established that the solutions containing Triton X-100 and sodium deoxycholate and the mixtures of these detergents with urea are the most effective. It was shown that the extraction of proteins from bacterial cells under the studied conditions is not accompanied by a release of DNA into solution but is associated with extraction of low-molecular RNAs. The level of protein extraction reaches 80%. No disruption of the bacterial cell wall occurs during the extraction, and proteins probably permeate through meshes of the murein network. The efficiencies of our buffer mixtures are close to or higher than that of the commercial reagent CelLytic B (Sigma, United States). The practical uses of the chaeotropic mixtures developed are discussed.

[Alternative splicing of pre-mRNA encoding the Musca domestica latrophilin-like protein(LLP): primary structures of four spliced forms of mRNA and their protein products].

An internal DNA fragment (approximately 2000 bp) homologous to the conserved regions of genes encoding latrophilin-like proteins (LLPs) was obtained by the PCR technique using degenerate primers to these gene regions. The gene-specific primers were synthesized based on the results of sequencing of the isolated fragment, and all overlapping cDNA fragments of the llp gene encoding the Musca domestica LLP were obtained by the rapid amplification of cDNA 5'- and 3'-ends (5'- and 3'-RACE). Four alternatively spliced mRNAs were found while sequencing the obtained cDNA fragments. Two long mRNAs (approximately 6000 nt) differ in the structures of both the sites encoding signal peptides and 5'-terminal untranslated regions. They encode large proteins (approximately 1800 aa), whose domain organization is similar to that of mammalian latrophilins. Each deduced protein contains a domain with seven transmembrane regions followed by an extended cytoplasmic C-terminal domain. Two other mRNA forms are derived from these long mRNAs; they encode proteins severly truncated at their C-termini (approximately 900 aa). They are composed of only three transmembrane regions and a short unique cytoplasmic C-terminal domain (23 aa). The limitations and drawbacks of the existing 3'-RACE techniques found during study of the long alternatively spliced cDNAs are analyzed, and ways for overcoming these difficulties are proposed.

[Dissociation of unstable cointegrate plasmids formed during transposition of IS1 element: the role of IS1 encoded recombinase]. / Dissotsiatsiia nestabil'nykh kointegratnykh plazmid, obrazuiushchikhsia pri transpozitsii élementa IS1: uchastie rekombinazy, kodiruemoi IS1.

The properties of IS1/Tn9'-mediated cointegrates between plasmids pDK57 (pBR322:: :: Tn9') and pRP3.1--the deletion derivative of RP1 were investigated. It was found that IS1/Tn9'-mediated integration of pDK57 into the active transcribed regions of pRP3.1 (in particular kan and tet genes) leads to formation of unstable cointegrates capable of resolving in E. coli K-12 rec+ and recA cells. The structure of dissociation products of unstable cointegrates was studied. According to the data received in rec+ cells, the unstable cointegrates mainly produced plasmids pDK57 and pBR322::IS1--Cms-derivative of pDK57 as resolution products. In recA cells the cointegrates dissociate in different ways, and this process leads to the formation of not only pDK57 and pBR322::IS1, but also to the production of the deletion derivatives of these plasmids as well as to the derivatives of pDK57 and pBR322::IS1, containing duplications of IS1 or separate parts of Tn9'. It was concluded that the IS1-specific recombinase is involved in the dissociation (resolution) of unstable IS1/Tn9'-mediated cointegrates. This recombinase recognizes the sites localized in both inverted termini of IS1 as well as in the adjacent DNA segments. Hence, it is possible, that the IS1 recombinase is involved also in the generation of IS1-adjacent delations.

[Structural instability of co-integrates formed during interaction of plasmid R57 with pB322 and RP1. Possible role of IS1 element in the degradation of co-integrates]. / Strukturnaia nestabil'nost' kointegratov, obrazuiushchiksia pri vzaimodeistvii plazmidy R57 s pBR322 i RP1. Vozmozhnoe uchastie élementa IS1 v degradatsii kointegratov.

The conjugative plasmid R57 determines resistance to ampicillin and chloramphenicol. Earlier it was shown that R57 encodes site-specific recA-independent recombinase, which acts in cis and resolves IS1-mediated cointegrates arising in the Escherichia coli recA cells between R57 and pBR322. In the present work the properties of the cointegrates between R57 and pBR322 or RP1 arising in the E. coli rec+ strains were studied. It was found that the cointegrates between R57 and pBR322, obtained by mating of the respective biplasmid donors of E. coli rec+ and the rec+ recipients, lost as a result of deletion a large DNA segment of R57 containing determinant Cmr. The resulting hybrid replicons preserved determinants Apr and Tcr of pBR322 and the R57 conjugative properties and were structurally identical. By using plasmid RP1ts12, which is temperature-sensitive in replication, it was demonstrated that in cells rec+ the cointegrates between R57 and RP1 are extremely unstable. On storage they undergo structural degradation mainly affecting the RP1 replicon. The degradation products of the hydrid complex had lost their RP1 genes but preserved the R57 functional determinants. For elucidation of the observed phenomena the properties of the IS1-mediated cointegrates between pBR322:Tn9 and plasmid pBR3.1--deletion derivative of RP1 were studied. It was found that insertion of IS1 sometimes resulted in formation of unstable cointegrates capable of resolving and loosing determinant Cmr with a high frequency. It was suggested that IS1 encodes the site-specific recombinase responsible for resolution of the IS1-mediated cointegrates and deletion generation. Expression of this recombinase appears to be dependent on structure of the insertion sites. The possible role of IS1 and recombinase encoded by it in resolution and structural instability of the cointegrates between R57 and pBR322 or RP1 is discussed.

[Immunity to repeated transposition of the insertion sequence IS21]. / Immunitet k povtornomu vstraivaniiu insertsionnoi posledovatel'nosti IS21.

The ability of pBR325 derivatives carrying a copy of IS21-element to accept the second copy of this element from plasmid pRP19.6, a temperature-sensitive for replication mutant of RPI containing the duplicated IS21 was studied. It was shown that the frequency of IS21 transposition into plasmids pBR32S::IS21 differing by localization IS21 was lower by two orders of magnitude as compared to that of pBR325. The restriction endonuclease analysis revealed that the insertion of the second copy of IS21 resulted in the formation of pBR325 derivatives carrying the tandem repeated copies of IS21. It was also shown that the plasmids pBR325::IS21 were capable of increasing the frequency of pRP19.6 insertion into the bacterial chromosome from 3-9 to 200-300 times depending on IS21 localization. On the basis of the results obtained and literature data the possible mechanism of the transposition immunity is discussed.

[Characteristic features of the structure of co-integrating plasmids and simple insertions formed during transposition of the IS1 element in transposon Tn9']. / Osobennosti struktury kointegratnykh plazmid i prostykh insertsii, obrazuiushchikhsia pri transpozitsii élementa IS1 v sostave transpozona Tn9'.

Earlier we have studied unstable dissociating IS1/Tn9'-mediated cointegrates between the plasmids pDK57 (pBR322::Tn9') and pRP3.1, a deletion derivative of RP1, and two types of such cointegrates containing three and four copies of IS1 were revealed. In the present paper we studied the structure of stable IS1/Tn9'-mediates cointegrates and simple insertions formed by interaction between the plasmids pDK57 and pRP3.1 in the E. coli recA- cells. It was shown, that the stable cointegrates were formed by insertion of pDK57 in different loci of pRP3.1 and these cointegrates contain three copies of IS1, i.e. one copy of IS1 and a copy of Tn9' at the junction of the two replicons. The cointegrates are formed predominantly due to the activity of the left copy of Tn9', which occupies a proximal position in regard to the promoter of the cat gene. It was found that the integration of pDK57 into the kan gene region of pRP3.1 leading to the formation of the KmS cointegrates occurs only in one of the two possible orientations. Meanwhile the insertions of the transposon Tn9' into the kan region of pRP3.1 leading to simple insertions occurs in the orientation opposite to the orientation of the transposon in the KmS cointegrates. It is proposed that simple insertions are not the products of direct transposition of Tn9', but they are formed from unstable cointegrates under the action of IS1-specific resolvase.

[Design of recombinant plasmids for effective Zymomonas mobilis pyruvate decarboxylase (pdk) gene expression in Bacillus subtilis cells]. / Konstruirovanie rekombinantnykh plazmid dlia éffektivnoi ékspressii gena piruvatdekarboksilazy (pdk) iz Zymomonas mobilis v kletkakh Bacillus subtilis.

The pdk gene from Z. mobilis localized on the 4.7-kb SpHI DNA fragment in plasmid pB201 was subcloned using DraI restriction endonuclease into the SmaI site of the phage cloning vector M13mp19. The derivatives of M13mp19 obtained, containing 1.8-kb inserts of the pdk gene in two opposite orientations, were used for DNA sequencing and site-directed mutagenesis. The latter was performed using polymerase chain reaction (PCR) and synthetic deoxyribonucleotides of appropriate structure as primers. In this way a BamHI site near the initial (formylmethionine) codon of the pdk gene was created. After amplification the pdk gene was treated by restriction endonuclease BamHI and cloned into pUC19, and then recloned into shuttle vector pCB20 capable of replicating in both Gram negative and Gram positive bacteria. A recombinant plasmid pCB20pdkI--a derivative of pCB20 carrying the pdk gene under control of the "expression unit" EU19035 containing a bacillar vegetative promoter and an RBS site was obtained. The properties of the pCB20pdkI in E. coli and Bac. subtilis cells were studied. It was shown that pCB20pdkI determines a high level of PDK synthesis in Bac. subtilis. At the same time, it strongly inhibits E. coli cell growth and segregates rapidly from this host.

[Interaction (integration and excision) of the pRP19.6 plasmid, a derivative of the RP1 plasmid containing duplicated sequence IS21, with the chromosome of Escherichia coli K12]. / Vzaimodeistvie (vstraivanie i iskliuchenie) plazmidy pRP19.6-proizvodnoi RP1, soderzhashchei duplitsirovannuiu posledovatel'nost' IS21, s khromosomoi Escherichia coli K12.

A pRP19.6 plasmid is the derivative of the temperature sensitive RPlts12 plasmid and contains a duplicated IS21 (IS8) element. Using temperature sensitive pRP19.6 replication, Hfr strains have been obtained by integration of the plasmid into the chromosome of E. coli rec+ and recA- cells and their properties were studied. According to the results obtained, pRP19.6 insertion into the genome of the rec+ bacteria IS reversible, and its integration into the chromosome of the recA- bacteria produced the stable Hfr strains. To elucidate the mechanism of pRP19.6 excision from the bacterial chromosome, plasmids of R+ transconjugates generated with a low frequency in the crosses between the stable Hfr strains and the rec+ recipients were analyzed. It was shown that the stable Hfr clones might produce stable R1 plasmids as well as a family of deletion KmsTra- derivatives of the pRP19.6. The structure of the KmsTra- was investigated and the mechanism of their formation was proposed. In the light of the data obtained, prospects of pRP19.6 practical application are discussed.

[Primary structure of two natural IS1-flanking transposons Tn9* and Tn9' coding for stability to chloramphenicol]. / Pervichnaia struktura dvukh prirodnykh IS1-flankirovannykh transposonov Tn9* i Tn9', kodiruiushchikh ustoichivost' k khloramfenicolu.

The DNA nucleotide sequence from the central region of the composite transposons Tn9* and Tn9' at the junction with the right copy of IS1 was determined. From the data obtained it follows that both transposons are members of the Tn9 family, although they contain additional DNA segments with regard to Tn9 of the length about 320 and 290 b.p. respectively lying distal to the cat gene. It was proposed that all the transposons of the Tn9 family have been formed as a result of IS1-mediated deletions of the plasmid R100 r-determinant. It was revealed from the data of computer analysis that in the sequenced DNA there are two potential promotors with transcription directed opposite in relation to the transcription of the cat gene.

[Thermosensitive vector derived from RP1 plasmid for detection of transposable elements]. / Termochuvstvitel'nyi vektor na osnove plazmidy RP1 dlia obnaruzheniia transpoziruemykh élementov.

The involvement of the transposable DNA element of E. coli K12 chromosome in integrative recombination of RP1 plasmid was studied. Using temperature sensitive for replication plasmid RP1ts12--the derivative of RP1 which contains mutated transposon Tnl, it was shown that integration of RP1 into host chromosome and Hfr formation may occur according to a mechanism mediated by chromosome IS-elements. Plasmids that are desintegrated from the chromosome of these Hfrs contain discrete DNA segments (IS-elements) and possess elevated frequency of integration into chromosome of rec+ cells. The latter was used for selection of RP1ts12 recombinants carrying chromosome IS. For identification of IS involved in RP1 integration the number of independent RP1ts 12 recombinants was subjected to restriction and heteroduplex analysis. By analysing recombinants integrated into bacterial chromosome with frequency 5 X 10(-3), a new IS-element of E. coli K12 designated IS111 was discovered. IS111-element is about 1500bp of length, contains Smal, Pst1 and BamH1 restriction endonuclease sites and was found in the same position on the plasmid RP1 in two different orientations. IS-elements that have been revealed in a number of other RP1ts12 recombinants were preliminary identified as IS1-like elements. One recombinants plasmid was found to have an IS5-like elements. The activity of IS-elements inserted into RP1ts12 in recA-dependent integrative recombination was estimated. From the data of absolute and relative RP1ts12 integration frequencies mediated by IS111, IS1- and IS5-like elements a conclusion was made about the absence of E. coli K12 chromosome IS-elements in RP1 plasmid. The Hfr-formation and chromosomal gene transfer by recombinant plasmids RP1ts12: IS111 were studied. The possibility to use insertion RP1ts12 derivatives for the estimation of copies number, mapping and definition of orientation of IS-elements in bacterial chromosome and the possibilities for detection of transposable DNA elements using RP1ts12 in a wide range of gram-negative bacteria are discussed.

[Structural and functional organization of the transposon Tn2555 carrying saccharose utilization genes]. / Strukturnaia i funktsional'naia organizatsiia transpozoma Tn2555, nesushchego geny utilizatsii sakharozy.

Transposon Tn2555 was isolated from a clinical E. coli strain carries the genes for sucrose utilization. Previously it was shown that Tn2555 is very unstable and undergoes structural rearrangements with a high frequency. Several deletion derivatives of Tn2555 and one with an inversion of the internal segment were found. They form the Tn2555 transposon family. This paper describes further structural and functional analysis of Tn2555. In the course of the experiments on pBR325 (Mob-) mobilization by conjugative RP4 derivatives, containing Tn2555 family elements, it was found, that all of them induce cointegrate formation. Some of these cointegrates were able to dissociate in rec+ and recA E. coli cells. Restriction endonuclease analysis of the resulting plasmids have shown, that among them were the end products of the Tn2555 transposition from RP4 to pBR325. Besides, the pBR325 derivatives, containing a discrete DNA segment of approximately 800 b.p., originating from Tn2555, were found. The segment can transpose from pBR325 to RP4 indicating that it is an insertion sequence. This new IS-element was designated IS286. The size and the genetic properties of IS286 resemble those of the IS1 element. However restriction analysis and Southern hybridization data show no significant homology between IS286 and IS1. It was found that the Tn2555 family elements are flanked by directly repeated IS286. One of them (Tn2555.3) contains an additional copy of IS286 in its internal region.

[Isolation and characteristics of a temperature-sensitive plasmid pRP19.6--an RP1 derivative containing the duplicated IS21 sequence]. / Poluchenie i kharakteristika termochuvstvitel'noi plazmidy pRP19.6--proizvodnoi RP1, soderzhashchei duplitsirovannuiu posledovatel'nost' IS21.

When analysing the antibiotic resistant, temperature-independent derivatives of Proteus mirabilis cells, carrying the plasmid RP1ts12, a derivative of the latter (pRP19.6) with an elevated frequency of integration into E. coli K12 chromosome, has been isolated. The structure and properties of pRP19.6 was studied. As revealed from the data of structural and genetic analyses pRP19.6 is identical to the factor R68.45 described earlier by Haas and Holloway. Similarly to R68.45, the plasmid under study contains two copies of IS21 sequence and mobilises nonconjugative plasmid pBR325 with high efficiency. Using the temperature sensitive replication of pRP19.6, frequency of it's integration into the chromosomes of E. coli rec+ and recA- stains is determined. It is demonstrated that the clones carrying the plasmid in integrated state are Hfr-strains. The possibilities to use the temperature sensitive R68.45 like plasmid for isolation of Hfr-strains in the broad range of gram-negative bacteria and for insertional inactivation of chromosomal genes are discussed.

[Study of the role of the insA open reading frame in the IS1 insertion element structure during transposition and resolution of the IS1 mediated cointegrates]. / Izuchenie roli ramki schityvaniia insA v strukture insertsionnogo élementa IS1 v protsessakh transpozitsii i razresheniia oposredovannykh IS1 kointegratov.

In order to elucidate the function of the IS1 insA gene derivatives of plasmid pUC19::Tn9' with insertions of synthetic oligonucleotides were obtained. The latter are equal or multiple of 9 b.p. in length and are located in the Pst1 site within each of the two IS1 copies of the Tn9' transposon. The insertions of the nine base oligonucleotides code for the neutral amino acids and do not shift the reading frame. One of the mutant transposon obtained - Tn9'/X was studied on the ability to form simple insertions and plasmid cointegrates. For this purpose the pUC19 derivatives carrying the wild type and mutant transposon were mobilized by conjugative plasmid pRP3.1. It was found that the damage of the insA gene does not influence the ability of transposon to form simple insertions and plasmid cointegrates in both recA - and rec+ cells of E. coli. However, the frequency of the cointegrate formation in the subsequent transposition of the mutant transposon from pRP3.1::Tn9'/X to pBR322 was by 10-20 times lower in comparison to the wild type transposon. Instable (dissociating) Tn9'/X-mediated plasmid cointegrates formed by interaction pUC19::Tn9'/X and pRP3.1 were obtained. It was shown that in the E. coli recA-cells such cointegrates dissociate, as a rule, "correctly", i.e. they segregate mainly plasmids of types pUC19::Tn9'/X and pUC19::IS1/X. The data obtained correspond with the notion that the gene insA product is not essential for transposition, but is, possibly, involved in the formation of the IS1-generated deletions.

[A new approach to the isolation of genomic DNA samples from yeast and fungi: preparation of DNA-containing cell envelopes and their use in PCR]. / Novyi podkhod dlia vydeleniia genomnoi DNK iz drozhzhei i gribov : poluchenie DNK-soderzhashchikh kletochnykh obolochek i ikh priamoe ispol'zovanie v PTsR.

A simple and rapid procedure for the preparation of yeast and fungal DNA samples useful in PCR amplification was developed. The DNA was purified from proteins, lipids, polysaccharides, and other impurities by their high-temperature extraction (in a boiling water bath) with buffer solutions containing chaotropic salts. Under these conditions, yeast and fungal cell envelopes remain unbroken and retain the original DNA and RNA that could be used for direct PCR amplification. We called the proposed PCR technique as the PCR using DNA-containing cell envelopes.

[The chromosomal genes for black widow spider neurotoxins do not contain introns]. / Khromosomnye geny neirotoksinov pauka karakurta ne soderzhat intronov.

The overlapping fragments of the chromosomal DNA from black widow spider Latrodectus mactans carrying genes for high-molecular-mass protein neurotoxins, alpha- and delta-latroinsectotoxins (alpha-LIT and delta-LIT) and alpha-latrotoxin (alpha-LTX), were PCR-amplified and cloned. Restriction analysis of the PCR products showed that the distribution and sizes of the restriction fragments coincided with those deduced from the earlier sequencing of cDNAs of the corresponding genes. It thus followed that the alpha-LIT and delta-LIT genes are intronless. Along with our data on the structure of the alpha-latrocrustotoxin (alpha-LCT), this implies that the lack of introns is a common feature of the black widow spider genes encoding high molecular mass neurotoxins.