Carbon nanotube linear bearing nanoswitches.

We exploit the remarkable low-friction bearing capabilities of multiwalled carbon nanotubes (MWNTs) to realize nanoelectromechanical switches. Our switches consist of two open-ended MWNT segments separated by a nanometer-scale gap. Switching occurs through electrostatically actuated sliding of the inner nanotube shells to close the gap, producing a conducting ON state. For double-walled nanotubes in particular, a gate voltage can restore the insulating OFF state. Acting as a nonvolatile memory element capable of several switching cycles, our devices are straightforward to implement, self-aligned, and do not require complex fabrication or geometries, allowing for convenient scalability.

Ballistic phonon thermal transport in multiwalled carbon nanotubes.

We report electrical transport experiments, using the phenomenon of electrical breakdown to perform thermometry, that probe the thermal properties of individual multiwalled carbon nanotubes. Our results show that nanotubes can readily conduct heat by ballistic phonon propagation. We determine the thermal conductance quantum, the ultimate limit to thermal conductance for a single phonon channel, and find good agreement with theoretical calculations. Moreover, our results suggest a breakdown mechanism of thermally activated C-C bond breaking coupled with the electrical stress of carrying approximately 10(12) A/m2. We also demonstrate a current-driven self-heating technique to improve the conductance of nanotube devices dramatically.

Novel bifunctional alkaline protease inhibitor: protease inhibitory activity as the biochemical basis of antifungal activity.

An alkaline protease inhibitor (API) from a Streptomyces sp. NCIM 5127 was shown to possess antifungal activity against several phytopathogenic fungi besides its antiproteolytic (anti-feedent) activity [J. V. Vernekar et al. (1999) Biochem. Biophys. Res. Commun. 262, 702-707]. Based on the correlation between antiproteolytic and antifungal activities in several tests such as copurification, heat inactivation, chemical modification, and its binding interaction with the fungal protease, we demonstrate, for the first time, that the dual function of API is a consequence of its ability to inhibit the essential alkaline protease. The parallel enrichment of both the functions during purification together with the heat inactivation of API leading to the concomitant loss of the two activities suggested their presence on a single molecule. Chemical modification of API with NBS resulted in the complete loss of antiproteolytic and antifungal activities, with no gross change in conformation implying the involvement of a Trp residue in the active site of the inhibitor and the presence of a single active site for the two activities. Treatment of API with DTT abolished both the activities although the native structure of API remained virtually unaffected, indicating the catalytic role of the disulfide bonds. Inactivation of API either by active site modification or by conformational changes leads to the concurrent loss of both the antiproteolytic and antifungal activities. Experimental evidences presented here serve to implicate that the antifungal activity of API is a consequence of its protease inhibitory activity.

Complexity in specificities and expression of Helicoverpa armigera gut proteinases explains polyphagous nature of the insect pest.

Helicoverpa armigera is a devastating pest of cotton and other important crop plants all over the world. A detailed biochemical investigation of H. armigera gut proteinases is essential for planning effective proteinase inhibitor (PI)-based strategies to counter the insect infestation. In this study, we report the complexity of gut proteinase composition of H. armigera fed on four different host plants, viz. chickpea, pigeonpea, cotton and okra, and during larval development. H. armigera fed on chickpea showed more than 2.5- to 3-fold proteinase activity than those fed on the other host plants. H. armigera gut proteinase composition revealed the predominance of serine proteinase activity; however, the larvae fed on pigeonpea revealed the presence of metalloproteases and low levels of aspartic and cysteine proteases as well. Gut proteinase activity increased during larval development with the highest activity seen in the fifth instar larvae which, however, declined sharply in the sixth instar. Over 90% of the gut proteinase activity of the fifth instar larvae was of the serine proteinase type, however, the second instar larvae showed the presence of proteinases of other mechanistic classes like metalloproteases, aspartic and cysteine proteases along with serine proteinase activity as evident by inhibition studies. Analysis of fecal matter of larvae showed significant increase in proteinase activity when fed on an artificial diet with or without non-host PIs than larvae fed on a natural diet. The diversity in the proteinase activity observed in H. armigera gut and the flexibility in their expression during developmental stages and depending upon the diet provides a base for selection of proper PIs for insect resistance in transgenic crop plants.

Characterization of acid-induced unfolding intermediates of glucose/xylose isomerase.

Acid-induced unfolding of the tetrameric glucose/xylose isomerase (GXI) from Streptomyces sp. NCIM 2730 has been investigated using intrinsic fluorescence, fluorescence quenching, second derivative spectroscopy, hydrophobic dye (1-anilino-8-naphthalene-sulfonate) binding and CD techniques. The pH dependence of tryptophanyl fluorescence of GXI at different temperatures indicated the presence of two stable intermediates at pH 5.0 and pH 3.0. The pH 3.2 intermediate was a dimer and exhibited molten globule-like characteristics, such as the presence of native-like secondary structure, loss of tertiary structure, increased exposure of hydrophobic pockets, altered microenvironment of tyrosine residues and increased accessibility to quenching by acrylamide. Fluorescence and CD studies on GXI at pH 5.0 suggested the involvement of a partially folded intermediate state in the native to molten globule state transition. The partially folded intermediate state retained considerable secondary and tertiary structure compared to the molten globule state. This state was characterized by its hydrophobic dye binding capacity, which is smaller than the molten globule state, but was greater than that of the native state. This state shared the dimeric status of the molten globule state but was prone to aggregate formation as evident by the Rayleigh light scattering studies. Based on these results, the unfolding pathway of GXI can be illustrated as: N-->PFI-->MG-->U; where N is the native state at pH 7.5; PFI is the partially folded intermediate state at pH 5.0; MG is the molten globule state at pH 3.2 and U is the monomeric unfolded state of GXI obtained in the presence of 6 M GdnHCl. Our results demonstrate the existence of a partially folded state and molten globule state on the unfolding pathway of a multimeric alpha/beta barrel protein.

Evidence for tryptophan in proximity to histidine and cysteine as essential to the active site of an alkaline protease.

The presence, microenvironment, and proximity of an essential Trp with the essential His and Cys residues in the active site of an alkaline protease have been demonstrated for the first time using chemical modification, chemo-affinity labeling, and fluorescence spectroscopy. Kinetic analysis of the N-bromosuccinimide- (NBS) or p-hydroxymercuribenzoate- (PHMB) modified enzyme from Conidiobolus sp. revealed that a single Trp and Cys are essential for activity in addition to the Asp, His, and Ser residues of the catalytic triad. Full protection by casein against inactivation of the enzyme by NBS and quenching of Trp fluorescence upon binding of the enzyme with NBS, substrate (sAAPF-pNA), or inhibitor (SSI) confirmed participation of the Trp residue at the substrate/inhibitor binding site of the alkaline protease. Comparison of the K(sv) values for the charged quenchers CsCI (1.66) and KI (7.0) suggested that the overall Trp microenvironment in the protease is electropositive. The proximity of Trp with His was demonstrated by the sigmoidal shape of the pH-dependent fluorometric titration curve with a pK(F) of 6.1. The vicinity of Trp with Cys was indicated by resonance energy transfer between the intrinsic fluorophore (Trp) and 5-iodoacetamide-fluorescein labeled Cys (extrinsic fluorophore). Our results on the proximity of Trp with essential His and Cys thus confirm the presence of Trp in the active site of the alkaline protease.

Pearl millet cysteine protease inhibitor. Evidence for the presence of two distinct sites responsible for anti-fungal and anti-feedent activities.

Recently, pearl millet cysteine protease inhibitor (CPI) was, for the first time, shown to possess anti-fungal activity in addition to its anti-feedent (protease inhibitory) activity [Joshi, B.N. et al. (1998) Biochem. Biophys. Res. Commun. 246, 382-387]. Characterization of CPI revealed that it has a reversible mode of action for protease inhibition. The CD spectrum exhibited a 35% alpha helix and 65% random coil structure. The intrinsic fluorescence spectrum was typical of a protein devoid of tryptophan residues. Demetallation of Zn2+ resulted in a substantial change in the secondary and tertiary structure of CPI accompanied by the complete loss of anti-fungal and inhibitory activity indicating that Zn2+ plays an important role in maintaining both structural integrity and biological function. The differential response of anti-fungal and inhibitory activities to specific modifiers showed that there are two different reactive sites associated with anti-fungal and anti-feedent activity in CPI located on a single protein as revealed from its N-terminal sequence data (AGVCYGVLGNNLP). Modification of cysteine, glutamic/aspartic acid or argnine resulted in abolition of the anti-fungal activity of CPI, whereas modification of arginine led to an enhancement of the inhibitory activity in solution. Modification of histidine resulted in a twofold increase in the protease inhibitory activity without affecting the anti-fungal activity, whereas modification of serine led to selective inhibition of the protease inhibitory activity. The differential nature of the two activities was further supported by differences in the temperature stabilities of the anti-fungal (60 degrees C) and inhibitory (40 degrees C) activities. Binding of papain to CPI did not abolish the anti-fungal activity of CPI, supporting the presence of two active sites on CPI. The differential behavior of CPI towards anti-fungal and anti-feedent activity cannot be attributed to changes in conformation, as assessed by their CD and fluorescence spectra. The interaction of CPI modified for arginine or histidine with papain resulted in an enhancement of CPI activity accompanied by a slight decrease in fluorescence intensity of 15-20% at 343 nm. In contrast, modification of serine resulted in inhibition of CPI activity with a concomitant increase of 20% in the fluorescence intensity when complexed by the enzyme. This implies the involvement of enzyme-based tryptophan in the formation of a biologically active enzyme-inhibitor complex. The presence of anti-fungal and anti-feedent activity on a single protein, as evidenced in pearl millet CPI, opens up a new possibility of raising a transgenic plant resistant to pathogens, as well as pests, by transfer of a single CPI gene.

Alkaline protease inhibitor: a novel class of antifungal proteins against phytopathogenic fungi.

A Streptomyces sp., which produces an alkaline protease inhibitor (API) exhibiting antifungal activity has been isolated from soil. The protein has been purified to homogeneity. The molecular characterization has revealed that it is a dimer (M(r) 28 kDa) with five disulphide linkages and has a pI of 3.8. API is a competitive type of inhibitor with a K(i) value of 2.5 x 10(-9) M. The inhibitor is stable over a pH range of 6 to 12 and a temperature range of 40 to 95 degrees C. API exhibits antifungal activity (in vitro) against phytopathogenic fungi such as Fusarium, Alternaria, and Rhizoctonia and also against Trichoderma, a saprophytic fungus. The antifungal activity of API appears to be associated with its ability to inhibit the fungal serine alkaline protease(s), which is indispensable for its growth. Retardation of the rate of fungal spore germination, as well as hyphal extention, was observed in the presence of API. Both the protease inhibitory and the antifungal activity were abolished on treatment of API with DTT (5 mM), suggestive of a common site for both the activities. This is the first report on API as a potential biocontrol agent against phytopathogenic fungi.

Diversity in inhibitors of trypsin and Helicoverpa armigera gut proteinases in chickpea (Cicer arietinum) and its wild relatives.

Developing seeds of eight chickpea (Cicer arietinum L.) cultivars (12-60 days after flowering) showed a significant variation in the trypsin inhibitor (TI) and the Helicoverpa armigera gut proteinase inhibitor (HGPI) content. For example, the highest TI (198 units/g) and HGPI (23 units/g) activities were exhibited by mature seeds of cv ICCV-2, whereas the lowest inhibitor activities were observed in cv PG8505-7 (96.1 TI units/g) and cv Vijay (5 HGPI units/g). Electrophoretic patterns showed a variation in TI bands during the early stages of seed development, indicating cultivar-specific TI accumulation. Among the seed organs, TI and HGPI activities were highly localized in the embryo-axis as compared to the cotyledons in immature and mature seeds. Moisture stress, as effected under rainfed conditions, resulted in reduced PI levels. Wild relatives of chickpea revealed variability in terms of the number and intensity of TI bands. However, when assessed for inhibition of HGP, none of the wild Cicer species showed more than 35% inhibition, suggesting that a large proportion of HGP was insensitive to PIs from Cicer. Our results provide a biochemical basis for the adaptation of H. armigera to the PIs of Cicer species and advocate the need for the transformation of chickpea with a suitable gene(s) for H. armigera resistance.

Molecular and biotechnological aspects of microbial proteases.

Proteases represent the class of enzymes which occupy a pivotal position with respect to their physiological roles as well as their commercial applications. They perform both degradative and synthetic functions. Since they are physiologically necessary for living organisms, proteases occur ubiquitously in a wide diversity of sources such as plants, animals, and microorganisms. Microbes are an attractive source of proteases owing to the limited space required for their cultivation and their ready susceptibility to genetic manipulation. Proteases are divided into exo- and endopeptidases based on their action at or away from the termini, respectively. They are also classified as serine proteases, aspartic proteases, cysteine proteases, and metalloproteases depending on the nature of the functional group at the active site. Proteases play a critical role in many physiological and pathophysiological processes. Based on their classification, four different types of catalytic mechanisms are operative. Proteases find extensive applications in the food and dairy industries. Alkaline proteases hold a great potential for application in the detergent and leather industries due to the increasing trend to develop environmentally friendly technologies. There is a renaissance of interest in using proteolytic enzymes as targets for developing therapeutic agents. Protease genes from several bacteria, fungi, and viruses have been cloned and sequenced with the prime aims of (i) overproduction of the enzyme by gene amplification, (ii) delineation of the role of the enzyme in pathogenecity, and (iii) alteration in enzyme properties to suit its commercial application. Protein engineering techniques have been exploited to obtain proteases which show unique specificity and/or enhanced stability at high temperature or pH or in the presence of detergents and to understand the structure-function relationships of the enzyme. Protein sequences of acidic, alkaline, and neutral proteases from diverse origins have been analyzed with the aim of studying their evolutionary relationships. Despite the extensive research on several aspects of proteases, there is a paucity of knowledge about the roles that govern the diverse specificity of these enzymes. Deciphering these secrets would enable us to exploit proteases for their applications in biotechnology.

Characterization of Helicoverpa armigera gut proteinases and their interaction with proteinase inhibitors using gel X-ray film contact print technique.

Since Helicoverpa armigera is a devastating pest, an attempt was made to separate its gut proteinases and assess their diversity. Gelatin coating present on the X-ray film was used as a substrate to detect electrophoretically separated proteinases of H. armigera gut extract on native polyacrylamide gel electrophoresis (PAGE), sodium dodecyl sulfate (SDS)-PAGE and isoelectric focusing gels. The method involves electrophoresis, followed by washing the gel with Triton X-100 in case of SDS-PAGE, equilibration of the gel in proteinase assay buffers, overlaying the gel on X-ray film followed by washing the film with hot water to remove hydrolyzed gelatin revealing bands of proteinase activity. Using this protocol, at least six different proteinase isoforms were detected in H. armigera gut contents while three isoproteinases were identified in a commercial bacterial proteinase preparation. Adoption of the technique facilitated characterization of the H. armigera gut proteinases (HGP) and provided an easy tool to study the properties of the individual proteinases without purification. The approximate molecular masses of HGP as determined by SDS-PAGE were: 172.9, 59.3, 54.9, 47.6, 44.1 and 41.6 kDa, and of bacterial proteinases: 180.7, 127.3 and 95.3 kDa. The isoelectric point (pI) values of HGP and bacterial proteinase were in the range of 5.1-7.1 and 3.5-7.7, respectively. Some of the HGP isoforms were found to be highly pH-sensitive and showed activity only at pH 10.0. The major HGPs were inhibited by phenylmethylsulfonyl fluoride but not by (4-amidinophenyl)-methanesulfonyl fluoride. Incubation of HGP-resolved electrophoretic gel strips in chickpea or winged bean proteinase inhibitor solution permitted identification of specific inhibitors of individual proteinases and revealed that the major HGPs were insensitive to chickpea inhibitors whereas winged bean inhibitors effectively inhibited all the HGPs. Our results suggest that considerable variability exists among the isoproteinases of H. armigera gut with respect to their pH optima and sensitivity towards chemical and plant proteinase inhibitors. Such diversity is of immense biological significance as it explains the polyphagous nature of the insect which imparts unique adaptability to it against the defensive proteinase inhibitors of its wide range of host plants.

Molecular cloning and expression of the glucose/xylose isomerase gene from Streptomyces sp. NCIM 2730 in Escherichia coli.

A partial genomic library of Streptomyces sp. NCIM 2730 was constructed in Escherichia coli using pUC8 vector and screened for the presence of the D-glucose/xylose isomerase (GXI) gene using an 18-mer mixed oligonucleotide probe complementary to a highly conserved six-amino acid sequence of GXI from actinomycetes. Eight clones which hybridized with the radiolabelled oligoprobe showed the ability to complement xylose isomerase-defective E. coli mutants. The restriction map of the insert from one (pMSG27) of the eight GXI-positive clones showing detectable GXI activity was constructed. GXI-deficient strains of E. coli were able to utilize xylose as the sole carbon source for their growth upon transformation with pMSG27. E. coli JM105 (pMSG27) and E. coli JC1553 (pMSG27) were inducible by IPTG suggesting that the expression of the cloned gene was under the control of the lacZ promoter. Western blot analysis revealed that the cloned gene is expressed as a fusion protein of M(r) 110. This is the first report of expression of a catalytically active GXI from Streptomyces in Escherichia coli.

Molecular and industrial aspects of glucose isomerase.

Glucose isomerase (GI) (D-xylose ketol-isomerase; EC. 5.3.1.5) catalyzes the reversible isomerization of D-glucose and D-xylose to D-fructose and D-xylulose, respectively. The enzyme has the largest market in the food industry because of its application in the production of high-fructose corn syrup (HFCS). HFCS, an equilibrium mixture of glucose and fructose, is 1.3 times sweeter than sucrose and serves as a sweetener for use by diabetics. Interconversion of xylose to xylulose by GI serves a nutritional requirement in saprophytic bacteria and has a potential application in the bioconversion of hemicellulose to ethanol. The enzyme is widely distributed in prokaryotes. Intensive research efforts are directed toward improving its suitability for industrial application. Development of microbial strains capable of utilizing xylan-containing raw materials for growth or screening for constitutive mutants of GI is expected to lead to discontinuation of the use of xylose as an inducer for the production of the enzyme. Elimination of Co2+ from the fermentation medium is desirable for avoiding health problems arising from human consumption of HFCS. Immobilization of GI provides an efficient means for its easy recovery and reuse and lowers the cost of its use. X-ray crystallographic and genetic engineering studies support a hydride shift mechanism for the action of GI. Cloning of GI in homologous as well as heterologous hosts has been carried out, with the prime aim of overproducing the enzyme and deciphering the genetic organization of individual genes (xylA, xylB, and xylR) in the xyl operon of different microorganisms. The organization of xylA and xylB seems to be highly conserved in all bacteria. The two genes are transcribed from the same strand in Escherichia coli and Bacillus and Lactobacillus species, whereas they are transcribed divergently on different strands in Streptomyces species. A comparison of the xylA sequences from several bacterial sources revealed the presence of two signature sequences, VXW(GP)GREG(YSTAE)E and (LIVM)EPKPX(EQ)P. The use of an inexpensive inducer in the fermentation medium devoid of Co2+ and redesigning of a tailor-made GI with increased thermostability, higher affinity for glucose, and lower pH optimum will contribute significantly to the development of an economically feasible commercial process for enzymatic isomerization of glucose to fructose. Manipulation of the GI gene by site-directed mutagenesis holds promise that a GI suitable for biotechnological applications will be produced in the foreseeable future.

Structure-function relationship of xylanase: fluorimetric analysis of the tryptophan environment.

Involvement of one out of three tryptophan residues in the active site of the low-molecular-mass xylanase from Chainia has been demonstrated previously [Deshpande, Hinge and Rao (1990) Biochim. Biophys. Acta 1041, 172-177]. The work described here aims at: (i) deducing the structure-function relationship for the tryptophan residue involved at the active site (a) by correlating the effect of N-bromosuccinimide (NBS) on the fluorescence and activity, and (b) by assessing the ability of xylan to protect against decrease in fluorescence versus activity of NBS-treated enzyme; and (ii) probing into the environment of the tryptophan residues by studying the quenching of their fluorescence by various solute quenchers in the presence and absence of guanidine hydrochloride (Gdn.HCl). Complete inactivation of the NBS-treated enzyme occurs well before the loss of fluorescence. Full protection by xylan (0.5%) of the inactivation of enzyme by NBS compared with 30% protection for the decrease in fluorescence confirms the participation of a single tryptophan at the substrate-binding site of the xylanase. The xylanase exhibited a rather low fluorescence emission maximum at 310 nm. There was no shift in the emission maximum on treatment of the enzyme with Gdn.HCl (6.5 M), indicating the rigidity of the microenvironment around tryptophan residues. The quenching studies with acrylamide suggested the occurrence of both collisional as well as static quenching processes. The enzyme retained full activity as well as the characteristic emission maximum at 310 nm in the presence of acrylamide (100 mM), indicating that quenching of fluorescence by acrylamide is a physical process. Acrylamide was more efficient as a quencher than CsCl or KBr. Treatment of the enzyme with Gdn.HCl resulted in an increase in accessibility of the quenchers to the fluorophore as suggested by an increase in the Stern-Volmer quenching constants (K(SV)) of the solute quenchers. The analysis of K(SV) and V values of KBr and CsCl suggests that the overall tryptophan microenvironment in the xylanase from Chainia is slightly electronegative.

Genome characterization of glucose-isomerase-producingStreptomyces sp. NCIM 2730: detection of sequences homologous to a rice repeat sequence.

Restriction analysis of the genomic DNA from a high glucose/xylose-isomerase-yieldingStreptomyces sp. NCIM 2730 revealed a number of distinct bands on a background smear, indicating the occurrence of repeated DNA sequences in the genome. Optical renaturation analysis indicated that 25% of the genome comprised rapidly reannealing sequences with a copy number of 50 and a kinetic complexity of 3×10(3). Hybridization of theStreptomyces genomic library with theStreptomyces DNA, supported the estimate of the repetitive DNA content derived from the re-association kinetics of the DNA. Hybridization of DNA from three differentStreptomyces species with a rice repetitive DNA probe revealed the presence of homologous sequences, which is a unique finding.

Cloning of promoter-active DNA sequences from Chainia (NCL 82.5.1) in Escherichia coli.

The ability of Escherichia coli cells to recognise and use the regulatory signals of genes from Chainia (NCL 82-5-1) was determined in vivo using gene fusions. DNA fragments from Chainia were cloned in E. coli using the promoter probe plasmid pJAC4. Four of the randomly selected recombinants exhibited varying strengths of promoter activity as assessed by the concentration of ampicillin required to kill 50% of the colonies (LD50 values) and by beta-lactamase activity. The origin of the inserts was confirmed by colony hybridization of clones with labelled genomic DNA of Chainia. The beta-lactamase activity of recombinant colonies was substantially higher (> 10-fold) than that of colonies transformed with pJAC4 without any insert. The results show that a few Chainia DNA sequences are recognized by E. coli as transcription initiation signals for the expression of beta-lactamase gene, inspite of the high guanine/cytosine content of the Chainia genome.

Evidence for specific interaction of guanidine hydrochloride with carboxy groups of enzymes/proteins.

Experimental evidence for the interaction of Gdn.HCl with carboxylate groups of the proteins is presented, for the first time, based on (i) the inhibition by low concentrations of Gdn.HCl of enzymes that are known to require essential carboxyl groups for their catalytic activity unaccompanied by structural changes in the protein and (ii) failure of the carboxyl-specific Woodward's reagent K to react specifically with the carboxyl groups of the proteins/enzymes pretreated with Gdn.HCl. The results demonstrate that the specificity and the reversibility of interaction of Gdn.HCl with carboxylate groups of proteins can be gainfully utilized for probing the functional role of carboxylate residues in the proteins.

Bacteriophage lambda as a cloning vector.

Extensive research has been directed toward the development of multipurpose lambda vectors for cloning ever since the potential of using coliphage lambda as a cloning vector was recognized in the late 1970s. An understanding of the intrinsic molecular organization and of the genetic events which determine lysis or lysogeny in lambda has allowed investigators to modify it to suit the specific requirements of gene manipulations. Unwanted restriction sites have been altered and arranged together into suitable polylinkers. The development of a highly efficient in vitro packaging system has permitted the introduction of chimeric molecules into hosts. Biological containment of recombinants has been achieved by introducing amber mutations into the lambda genome and by using specific amber suppressor hosts. Taking advantage of the limited range of genome size (78 to 105% of the wild-type size) for its efficient packaging, an array of vectors has been devised to accommodate inserts of a wide size range, the limit being 24 kbp in Charon 40. The central dispensable fragment of the lambda genome can be replaced by a fragment of heterologous DNA, leading to the construction of replacement vectors such as Charon and EMBL. Alternatively, small DNA fragments can be inserted without removing the dispensable region of the lambda genome, as in lambda gt10 and lambda gt11 vectors. In addition, the introduction of many other desirable properties, such as NotI and SfiI sites in polylinkers (e.g., lambda gt22), T7 and T3 promoters for the in vitro transcription (e.g., lambda DASH), and the mechanism for in vivo excision of the intact insert (e.g., lambda ZAP), has facilitated both cloning and subsequent analysis. In most cases, the recombinants can be differentiated from the parental phages by their altered phenotype. Libraries constructed in lambda vectors are screened easily with antibody or nucleic acid probes since several thousand clones can be plated on a single petri dish. Besides the availability of a wide range of lambda vectors, many related techniques such as rapid isolation of lambda DNA, a high efficiency of commercially available in vitro packaging extracts, and in vitro amplification of DNA via the polymerase chain reaction have collectively contributed to lambda's becoming one of the most powerful and popular tools for molecular cloning.

Molecular cloning and expression of the xylanase gene from Chainia in Escherichia coli.

A complete genomic library of Chainia was constructed in coliphage lambda vector gt10 and was screened for the xylanase gene using an 18-mer mixed oligonucleotide probe corresponding to a six-amino acid sequence of low molecular mass Chainia xylanase. Inserts from 11 putative clones, showing hybridization with the oligonucleotide probe at medium stringency, were subcloned in pUC8 and screened for xylanase gene expression using anti-xylanase antibodies. The restriction map of the insert (1.4 kb) from one of the four immunopositive clones (PVX8) showing detectable xylanase activity was constructed. The xylanase activity of PVX8 was not induced by IPTG or xylan. Reorientation of the insert by directional cloning into pUC9 had no effect on the xylanase activity suggesting that an indigenous promoter from Chainia is responsible for the xylanase activity.