Site-directed mutagenesis to identify key residues in structure-function relationship of winged bean chymotrypsin-trypsin inhibitor and 3-D structure prediction

Winged bean chymotrypsin trypsin inhibitor (WbCTI) is a Kunitz type serine protease inhibitor that inhibits both trypsin and chymotrypsin at 1:1 molar ratio. Site-directed mutagenesis study was employed to generate two mutants of WbCTI, with an aim to explore its dual inhibitory properties against the proteases. The mutants were expressed in Escherichia coli and, were purified to homogeneity using a single step immunoaffinity column. The two mutants, each containing a single mutation at the amino acid sequence positions of 63 and 64, were named as L63A and R64A, respectively. Purified L63A-WbCTI exhibited anti-trypsin activity with no anti-chymotrypsin activity whereas R64A-WbCTI could inhibit chymotrypsin but not trypsin. To investigate the binding interactions between the mutated forms of WbCTI with the putative proteases, binding studies were carried out using gel filtration chromatography which further confirmed the formation of enzyme-inhibitor complexes. Finally, 3D model structure of WbCTI was designed using computer simulations which further emphasize the roles of L63 and R64 residues for dual inhibitory characteristics of WbCTI.     

Characterisation of the beta-tubulin gene of Cylicocyclus nassatus

mRNA and genomic DNA were isolated from adult Cylicocyclus nassatus, and the mRNA was reverse transcribed. The cDNA was PCR amplified using degenerate primers designed according to the alignment of the β-tubulin amino acid sequences of other species. To complete the coding sequence, the 3′ end was amplified with the 3′-RACE, and for amplification of the 5′ end the SL1-primer was used. The cDNA of the β-tubulin gene of C. nassatus spans 1429 bp and encodes a protein of 448 amino acids. Specific primers were developed from the cDNA sequence to amplify the genomic DNA sequence and to analyse the genomic organisation of the β-tubulin gene. The complete sequence of the genomic DNA of the β-tubulin gene of C. nassatus has a size of 2652 bp and is organised into nine exons and eight introns. The identities with the exons of the gru-1 β-tubulin gene of Haemonchus contortus range between 79% and 97%.     

Isolation of a root-specific cDNA encoding a ns-LTP-like protein from the roots of bean (Phaseolus vulgaris L.) seedlings

A root-specific cDNA clone, PVR3, was isolated from a bean (Phaseolus vulgaris L.) root cDNA library by a differential screening procedure. The nucleotide sequence of PVR3 contains an open reading frame coding for an 11.14 kDa polypeptide of 102 amino acid residues; the first 25 amino acids correspond to the sequence characteristic of a signal peptide. Comparison of the deduced PVR3 polypeptide sequence with the polypeptide sequences of previously cloned genes indicates that PVR3 may encode a ns-LTP-like protein. Molecular modelling of the PVR3 protein predicts that it has a three-dimensional structure that is similar to the three-dimensional model determined from the maize ns-LTP. The PVR3 mRNA accumulated mainly in the roots of young seedlings. It can be detected at low levels in flowers, but it is not detected in other organs. Genomic Southern blot analysis indicates that the genomic DNA corresponding to PVR3 cDNA is encoded by a single gene or small gene family in the bean genome.     

Narbonin,a novel 2S protein from Vicia narbonensis L. seeds: cDNA,gene structure and developmentally regulated formation

cDNA and genomic clones encoding narbonin, a 2S globulin from the seed of narbon bean (Vicia narbonensis L.), were obtained using the polymerase chain reaction (PCR) and sequenced. The full-length cDNA as well as genomic clones contain a single open reading frame (ORF) of 873 bp that encodes a protein with 291 amino acids comprising the mature narbonin polypeptide (M _r ca. 33 100) and an initiation methionine. The deduced amino acid sequence lacks a transient N-terminal signal peptide. The genomic clones do not contain any intron. No homology was found to nucleic acid and protein sequences so far registered in sequence data libraries. The biosynthesis of narbonin during embryogenesis is developmentally-regulated and its pattern of synthesis closely resembles that of typical seed storage globulins. However, during seed germination narbonin was degraded very slowly, indicating that it may have other function than storage protein. Southern analysis suggests the existence of a small narbonin gene family. Narbonin genes were also found in four different species of the genus Vicia as well as in other legumes such as Canavalia ensiformis and Glycine max. In Escherichia coli a recombinant narbonin was produced which yielded crystals like those prepared from narbonin purified from seeds.     

Genomic and cDNA cloning, characterization of Delonix regia trypsin inhibitor (DrTI) gene, and expression of DrTI in Escherichia coli

Degenerate primers were designed based on all possible sequences of the N-terminal and C-terminal regions of Delonix regia trypsin inhibitor (DrTI). Five hundred sixty-one bp of polymerase chain reaction (PCR) product was amplified using the above degenerate primers and genomic DNA and cDNA of Delonix regia as a template. The amplified PCR products were cloned and sequenced. DNA sequence analysis of cDNA and genomic clones of DrTI have the same nucleotide sequence in the coding region, and manifested a genomic clone without intervening sequences in the coding region. The amino acid sequence deduced from the DrTI genomic and cDNA clones agreed with that identified via amino acid sequencing analysis, except that two amino acid residues, Ser and Lys, existed between residues Lys141 and Ser142. DrTI open reading frame was then amplified and cloned in-frame with GST in pGEX4T-1 and overexpressed in Escherichia coli to yield a glutathione S-transferase (GST)-fusion protein with a calculated molecular mass of about 45 kDa. The recombinant DrTI (reDrTI) was derived by treating the GST-DrTI fusion protein with thrombin. Both the reDrTI and GST-DrTI fusion protein exhibited a strong identical inhibitory effect on trypsin activity.     

Identification of Cu/Zn superoxide dismutase in cattle and river buffaloes

All air-living organisms produce superoxide dismutase (SOD) and several antioxidant enzymes that limit oxidative stress by detoxifying reactive oxygen species. SOD1 gene has been investigated in Egyptian native cattle and buffalos at the level of genomic DNA and cDNA that were extracted from leucocytes. An unexpected band at approximately 370 bp was obtained in cattle genomic DNA and cDNA as well as in buffalo cDNA. SOD1 amplified sequence of native cattle genomic DNA and cDNA showed a 93% alignment. Native cattle genomic DNA SOD1 amplicon shares sequence homology with mRNAs of Bos taurus “similar to superoxide dismutase” (SOD1) sequence of the GeneBank database. It also shares sequence homology with “similar to superoxide dismutase” on B. taurus chromosome BTA13. The results indicate that the genomic DNA of Egyptian native cattle contains SOD1 processed pseudo gene. SOD1 primers amplified three fragments in buffalo genomic DNA which indicates that buffalo genome has different copies of SOD1 due to alternative splicing. It failed to produce the 370 bp fragments found in cattle DNA. The protein analysis revealed no differences between Egyptian native cattle and B. taurus SOD1 mRNA. However, one amino acid, aspartic acid (Asp), in Egyptian native cattle and B. taurus SOD1, is substituted with asparagine (Asn) (D26N) in buffaloes. This amino acid substitution may be due to non-synonymous single nucleotide polymorphisms (nsSNPs). The nsSNPs detected in buffaloes may affect the function of the encoded protein. This study is the first investigation reporting that the resistance of the buffalo to diseases and parasites that afflict cattle may not be acquired but may have a genetic basis.     

Sequence analysis,cloning and over-expression of an endoxylanase from the alkaliphilic <Emphasis Type="BoldItalic">Bacillus halodurans</Emphasis>

The BhMIR32 xyn11A gene, encoding an extracellular endoxylanase of potential interest in bio-bleaching applications, was amplified from Bacillus halodurans MIR32 genomic DNA. The protein encoded is an endo-1,4-β-xylanase belonging to family 11 of glycosyl hydrolases. Its nucleotide sequence was analysed and the mature peptide was subcloned into pET22b(+) expression vector. The enzyme was over-expressed in a high density Escherichia coli culture as a soluble and active protein, and purified in a single step by immobilised metal ion affinity chromatography with a specific activity of 3073 IU mg⁻¹.     

Genetic analysis of cytochrome b5 from arachidonic acid-producing fungus, Mortierella alpina 1S-4: cloning, RNA editing and expression of the gene in Escherichia coli, and purification and characterization of the gene product.

Information on the amino acid sequences of the internal peptide fragments of cytochrome b5 from Mortierella hygrophila was used to prepare synthetic oligonucleotides as primers for the polymerase chain reaction. A 100-base DNA fragment was thus amplified, by using a genomic gene from Mortierella alpina 1S-4 as a template, which produced polyunsaturated fatty acids such as arachidonic acid. The amplified DNA fragment was used as the probe to clone both a 523-base cDNA fragment and a 2.1-kilobase SalI-NruI genomic fragment coding for the whole M. alpina 1S-4 cytochrome b5. On the basis of nucleotide sequences of both cytochrome b5 genomic gene and cDNA, the genomic cytochrome b5 gene was found to consist of four exons and three introns. A novel type of RNA editing, in which the cDNA included either guanine insertion or adenine-->guanine substitution at one base upstream of poly(A), was interestingly observed. The deduced amino acid sequence of M. alpina 1S-4 cytochrome b5 showed significant similarities with those of cytochrome b5s from other organisms such as rat, chicken, and yeast. The soluble form of the cytochrome b5 gene was expressed to 16% of the total soluble protein in Escherichia coli. The holo-cytochrome b5 accounted for 8% of the total cytochrome b5 in the transformants. The purified cytochrome b5 showed the oxidized and reduced absorbance spectra characteristic of fungal microsomal cytochrome b5.     

The flavin-containing monooxygenase expressed in pig liver: primary sequence, distribution, and evidence for a single gene

The primary sequence of the flavin-containing monooxygenase expressed in pig liver has been derived from the nucleotide sequence of cloned cDNA. The derived sequence is composed of 532 amino acids and represents a protein having a molecular weight of 58,952. The complete sequence was obtained from a single clone containing 2070 bases. A second clone, obtained from an independent library, yielded an identical sequence for the 1374 bases present. The amino acid composition compiled from the derived sequence is very similar to that obtained previously from the purified protein. In addition, a 10 amino acid sequence in a peptide formed from the purified protein by digestion with V8 protease exactly matches the derived sequence for residues 309-318. The flavin-containing monooxygenase expressed in pig liver is also expressed in pig lung and kidney as determined by analysis of both microsomal proteins and mRNA. The ratio of mRNA to protein for the enzyme in kidney is about 5 times greater than the same ratio for liver and about twice the ratio for lung. The reasons for these differences are not understood. Southern analysis of genomic DNA indicates that there is a single gene encoding the flavin-containing monooxygenase expressed in pig liver. Therefore, the broad activity of this enzyme in liver appears to be the result of the catalytic diversity of a single protein.     

Characterisation of the beta-tubulin gene of Cylicocyclus nassatus?

mRNA and genomic DNA were isolated from adult Cylicocyclus nassatus, and the mRNA was reverse transcribed. The cDNA was PCR amplified using degenerate primers designed according to the alignment of the β-tubulin amino acid sequences of other species. To complete the coding sequence, the 3′ end was amplified with the 3′-RACE, and for amplification of the 5′ end the SL1-primer was used. The cDNA of the β-tubulin gene of C. nassatus spans 1429 bp and encodes a protein of 448 amino acids. Specific primers were developed from the cDNA sequence to amplify the genomic DNA sequence and to analyse the genomic organisation of the β-tubulin gene. The complete sequence of the genomic DNA of the β-tubulin gene of C. nassatus has a size of 2652 bp and is organised into nine exons and eight introns. The identities with the exons of the gru-1 β-tubulin gene of Haemonchus contortus range between 79% and 97%.     

Gene structure,cDNA sequence,and developmental regulation of a low molecular weight hemolymph protein from Locusta migratoria

From a Locusta migratoria genomic DNA library, a gene has been isolated that codes for a previously unrecognized hemolymph protein of M_r = 19,000, designated 19k protein. The gene has at least five exons, extending over about 9 kb of DNA. Its polypeptide product, obtained by cell-free translation of mRNA selected from adult fat body RNA by hybridization with the cloned DNA, is precipitated by antiserum against a low molecular weight hemolymph protein fraction. The mature protein product has been purified from locust hemolymph, and an N-terminal sequence of 20 amino acids has been determined. In polyacrylamide gel electrophoresis, this protein comigrates with apolipophorin III, from which it was previously not distinguished, but it is clearly distinct by amino acid composition and sequence. The genomic clone was used as a probe to isolate a fat body cDNA clone of the 19k protein mRNA. The 938-base pair cDNA clone contains a 516-base pair open reading frame. The deduced 172-amino acid polypeptide includes an apparent signal peptide, a sequence of four amino acids that may represent a prosegment, and a sequence identical (with a single exception, which may reflect polymorphism) with the N-terminal sequence of the hemolymph protein. Its mRNA occurs at a low level in late larval fat body, is abundant in the newly eclosed adult, then declines to a low level, and rises again at days 8–10; it is greatly reduced after destruction of the corpora allata with precocene and then is elevated after treatment with methoprene, suggesting stimulation by juvenile hormone. The biological role of 19k protein is unknown.     

MEI1, an Arabidopsis gene required for male meiosis: isolation and characterization

 The T-DNA tagged mutant gene of Arabidopsis thaliana, mei1, produces after meiosis an abnormal tetrad, consisting of five to eight microspores of varying sizes and DNA contents. Plant DNA flanking the inserted T-DNA was isolated by inverse PCR. An approximately 16-kb DNA fragment spanning the T-DNA insertion site was isolated by screening a wild-type genomic library, using the plant flanking DNA as a probe. Using RT-PCR and RNA isolated from very young flower buds, a cDNA fragment was obtained. Nucleotide sequence comparison of the cDNA and the genomic sequence in this region indicated a gene which contained two introns. The 5′ and 3′ splice sites of neither intron comply with the :GU...AG: rule. In the mutant, the T-DNA had inserted into one of the introns. The deduced sequence of the MEI1 wild-type gene, which contains 89 amino acids, shows possible similarity with the human acrosin-trypsin inhibitor, HUSI-II, and is about the same size. Two wild-type DNA fragments, both extending over the T-DNA insertion site, were introduced into mutant plants by Agrobacterium-mediated transformation and plants were selected for both hygromycin and kanamycin resistance. Several independent male-fertile transformants were obtained with one of the DNA fragments. The fragment showing complementation of the mutant phenotype indicated that the sequence with similarity to the acrosin-trypsin inhibitor is MEI1. Within the 16-kb genomic fragment two other genes were identified; one showed no overall similarity to any protein sequence in the database and the other had almost complete identity with an Arabidopsis-transcribed sequence tag with similarity to ACC oxidase. Double mutants between mei1 and qrt1 were made, permitting better characterization of the mei1 phenotype because the individual microspores continued to be held together after callose dissolution. Received: 21 April 1998 / Revision accepted: 11 June 1998     

Purification and molecular characterization of a soybean polygalacturonase-inhibiting protein

A polygalacturonase-inhibiting protein (PGIP) was detected in soybean (Glycine max (L.) Merr.) seedlings. The protein was purified from germinating seeds and appeared to consist of at least three components with very close molecular weights (between 37 and 40 kDa) but each showing a unique N-terminal sequence. Primers specific for N-terminal and C-terminal nucleotide sequences of field bean (Phaseolus vulgaris L.) PGIP were used in a polymerase chain reaction (PCR) on soybean DNA, and only one amplification band was obtained. The amplified product was cloned and one of the PCR clones was sequenced. The nucleotide sequence comprises 942 bp with a single open reading frame which encodes a polypeptide of 313 amino-acid residues with a predicted molecular weight of 33984 Daltons and an isoelectric point of 8.21. Analysis of genome organization showed a single gene copy of PGIP with few related sequences, and wounding of soybean hypocotyls showed a strong induction of expression of the PGIP gene. The PGIP showed different activities toward three purified fungal endo-polygalacturonases (endo-PGs) (two endoPGs from Sclerotinia sclerotiorum and one endo-PG from Aspergillus niger). A possible involvement of soybean PGIP in plant defence against fungal pathogens is discussed.     

Cloning and Sequencing of the xynA Gene Encoding Xylanase A of Aspergillus kawachii

We have cloned the xynA gene coding for xylanase A, a major component of the xylanase family, from Aspergillus kawachii. The cDNA was isolated from an A. kawachii cDNA library by immunoscreening using antibody raised against the purified xylanase A protein. Nucleotide sequence analysis of the cDNA showed a 981-bp open reading frame that encoded a protein of 327 amino acid residues. The signal peptide was composed of 25 amino acid residues and the N-terminus of the mature protein was pyroglutamic acid. The transformed yeast with a cloned cDNA produced xylanase. The genomic DNA was arranged as ten exons and nine introns.     

The At4g11330 (ATMPK5) locus of<Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>: Updating the annotation

The completed sequence of theArabidopsis genome shows that a single base pair deletion exists in the gene encoding the MAP kinase ATMPK5 (locus At4g11330) when compared with the cDNA. This has led to the annotation of ATMPK5 in databases as a truncated protein kinase or with a deletion in kinase subdomain I. Both situations would lead to a mutated nonfunctional protein. We amplified and sequenced the relevant region from genomic DNA of theArabidopsis ecotypes Columbia and Wassilewskija, and we found that both contain sequences that are in accordance with the cDNA sequence. We propose, therefore, that the ATMPK5 locus can encode a functional MAP kinase protein. This finding is relevant to the further functional analysis of this locus.