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PubMed1987

Noradrenaline turnover after left coronary artery ligation in rat heart.

The levels and the turnover of noradrenaline were measured in the left and right ventricles following left coronary artery ligation for 2 h in untreated, adrenalectomized or hexamethonium-treated rats. Noradrenaline in the left ventricles was decreased by ligation and unaffected in the right ventricles, whether of untreated, adrenalectomized or hexamethonium-treated animals. Turnover in the left ventricles, as estimated by the decrease of noradrenaline under dopamine beta-hydroxylase inhibition with bis-(4-methyl-1-homo-piperazinyl-thiocarbonyl)-disulfide (FLA-63), was unaffected by ligation whether in untreated, adrenalectomized, or hexamethonium-treated rats. Ligation accelerated turnover in the right ventricles of untreated rats or attenuated it in adrenalectomized rats and caused no changes in hexamethonium-treated rats. These results suggest that noradrenaline turnover in ischemic myocardium is not affected by regional ischemia and that such ischemia might accelerate turnover in the non-ischemic area, probably as a result of increased activity of sympatho-adrenal reflexes.

Adrenalectomy↗
PubMed1985

Modification of the amino acid acceptor stem of E. coli tRNAMetf by ligation of chemically synthesized ribooligonucleotides.

The single-stranded region of the amino acid acceptor stem corresponding to the 3'-end of E. coli tRNAMetf was replaced by ligation of chemically synthesized ribooligonucleotides, in order to change the length of the single-stranded CCA terminus. The chemically synthesized ribooligomers, CCA, ACCA, AACCA and CAACCA, were ligated to nuclease-treated E. coli tRNAMetf, which lacked the ACCA sequence at the 3'-end. The methionine acceptor activities of these modified tRNAs were examined using E. coli methionyl-tRNA synthetase. Ligation of the chemically synthesized pentamer (AACCA) to the acceptor terminus restored the methionine acceptor activity, whereas ligation of the hexamer (CAACCA) or trimer (CCA) to the acceptor terminus did not Modification of the acceptor terminus had no effect on the formylation of accepted methionine.

Amino Acids↗
PubMed1996

Postpartum tubal ligation after pregnancy complicated by preeclampsia or gestational hypertension.

OBJECTIVE: To determine the anesthetic and surgical morbidity associated with postpartum tubal ligation after pregnancy complicated by pregnancy-induced hypertension. METHODS: Preoperative hemodynamic measurements, laboratory results, choice of anesthetic technique, intraoperative hemodynamic changes, and postoperative morbidity were compared in 53 women with pregnancy-induced hypertension (hypertensive group) and 53 controls who underwent postpartum tubal ligation between October 1992 and November 1995. We used a retrospective case-control design. RESULTS: Preoperative mean blood pressure (BP) measurements ( +/- standard deviation) were greater in hypertensive women than in controls (158 +/- 22/91 +/- 12 versus 126 +/- 13/71 +/- 10 mmHg; P < .001). Among women given spinal anesthetics for tubal ligation, the minimum intraoperative systolic BP was significantly lower in controls than in hypertensive women (P < .05). However, the maximum percentage decrease in systolic BP was greater in hypertensive women than in controls (33 +/- 14 versus 22 +/- 10%; P < .05). Only one patient in each group developed intraoperative hypertension. The percentage of patients discharged later than the first postoperative day was greater in hypertensive women than in controls (23 versus 8%; P < .05). CONCLUSION: The lack of profound hemodynamic responses during spinal or general anesthesia for postpartum tubal ligation supports the continued use of this procedure in selected women with pregnancy-induced hypertension.

Female↗
PubMed1992

Nucleotide sequencing of 5' and 3' termini of bovine viral diarrhea virus by RNA ligation and PCR.

Genomic RNA was extracted from cytopathic (CP) bovine viral diarrhea virus (BVDV) strain NADL, CP strain 72, and noncytopathic (NCP) strain SD-1 purified by ultracentrifugation. Assuming the presence of a cap structure, de-blocking of the 5' capped end of the genomic RNA was done by treatment with tobacco acid pyrophosphatase (TAP). Following decapping, the RNA molecules were ligated using T4 RNA ligase and the ligated tandem RNA templates were then amplified by primer-directed amplification (PCR). cDNA synthesis was done using reverse transcriptase with random primers and cDNA amplification was done using a negative sense primer 231-248 and a positive sense primer 12434-12451. The nucleotide sequence of the amplified product was determined by double-stranded sequencing using the Sanger di-deoxy chain termination method and an additional 'CCCCC' nucleotide sequence was identified at the ligation site. Following dATP tailing of cDNA and amplification across the 5' terminus and nucleotide sequencing, no additional nucleotides were identified on the 5' terminus. The 5' terminus as published by Collett et al., 1988b was confirmed as previously reported. Therefore, the 3' terminus includes an additional 'CCCCC' nucleotide sequence to that previously reported. Identical results were obtained when the BVDV genomic RNA was not decapped prior to RNA ligation and amplification.

Animals↗
PubMed1996

Application of linker-ligation-PCR for construction of phage display epitope libraries.

An efficient method for construction of random epitope libraries using filamentous phage is described. Random DNA fragments generated by DNase I digestion were blunt ended by T4 DNA polymerase and ligated with a 12-mer linker, followed by PCR amplification using the same oligonucleotide linker as primers. The results showed that only the ligated product containing linker sequences on both ends was specifically amplified. When the linker ligated-PCR (LL-PCR) product was used for the construction of phage display epitope libraries, the total number of independent clones in the libraries was increased by 100 to 1000 fold in comparison to that obtained for libraries constructed using other methods. In addition, the LL-PCR strategy also increased the probability of isolating recombined DNA fragments which are derived by random in-frame ligation of two or more discontinuous peptide-coding sequences before being inserted into the display vector. Such randomly recombined DNA fragments might be useful in defining conformational epitopes.

African Swine Fever Virus↗
PubMed1983

Ligation of highly modified bacteriophage DNA.

After digestion by TaqI or nicking by DNAase I, five highly modified bacteriophage DNAs were tested as substrates for T4 DNA ligase. The DNAs used were from phages T4, XP12, PBS1, SP82, and SP15, which contain as a major base either glucosylated 5-hydroxymethylcytosine, 5-methylcytosine, uracil, 5-hydroxymethyluracil, or phosphoglucuronated, glucosylated 5-(4',5'-dihydroxypentyl)uracil, respectively. The relative ability of cohesive-ended TaqI fragments of these DNAs and of normal, lambda DNA to be ligated was as follows: lambda DNA = XP12 DNA greater than SP82 DNA approximately equal to nonglucosylated T4 DNA greater than T4 DNA = PBSI1 DNA much greater than SP15 DNA. Taq I-T4 DNA fragments were also inefficiently ligated by Escherichia coli DNA ligase. However, annealing-independent ligation of DNAase I-nicked T4, PBS1, and lambda DNAs was equally efficient. We conclude that the poor ligation of Taq I fragments of T4 and PBS1 DNAs was due to the hydroxymethylation (and glucosylation) of cytosine residues at T4's cohesive ends and the substitution of uracil residues for thymine residues adjacent to PBS1's cohesive ends destabilizing the annealing of the restriction fragments. Only SP15 DNA with its negatively charged, modified base was unable to serve as a substrate for T4 DNA ligase in an annealing-independent reaction; therefore, its modification directly interfered with enzyme binding or catalysis.

Bacteriophages↗
PubMed1991

Electron paramagnetic resonance investigations of exogenous ligand complexes of low-spin ferric chloroperoxidase: further support for endogenous thiolate ligation to the heme iron.

Previous spectroscopic studies of chloroperoxidase have provided evidence for endogenous thiolate sulfur donor ligation to the central heme iron of the enzyme. This conclusion is further supported by recent DNA sequence data which revealed the existence of a third cysteine residue (in addition to a disulfide pair detected earlier) in the protein available for coordination to the heme iron. Thus, chloroperoxidase shares many spectroscopic properties with cytochrome P-450, the only other known thiolate-ligated heme protein. Surprisingly, a previous electron paramagnetic resonance (EPR) study of low-spin ferric chloroperoxidase-ligand complexes (Hollenberg, P.F., Hager, L.P., Blumberg, W.E. and Peisach, J. (1980) J. Biol. Chem. 255, 4801-4807) was unable to provide clear support for the presence of a thiolate ligand, although sulfur coordination was implicated. This was, in part, because an insufficient number of complexes was examined. In this work, we have significantly expanded upon the previous EPR study by using an extensive variety of over twenty exogenous ligands including carbon, nitrogen, oxygen, phosphorus and sulfur donors. Crystal field analysis, using the procedure of Blumberg and Peisach, of the present data in comparison with data for analogous complexes of cytochrome P-450-CAM, thiolate-ligated heme model systems, and myoglobin, is clearly indicative of endogenous thiolate ligation for chloroperoxidase. In addition, the UV-visible absorption and EPR spectral data suggest that a carboxylate ligand is a possible candidate for the endogenous sixth ligand to the heme iron that is responsible for the reversible conversion of ferric chloroperoxidase from high-spin to low-spin at low temperatures (less than 200 K).

Chloride Peroxidase↗
PubMed1995

A chemical modification of cytochrome-c lysines leading to changes in heme iron ligation.

Although 13 lysines of horse cytochrome c are invariant, and three more are extremely conserved, the modification of their side-chain epsilon-amino groups by beta-thiopropionylation caused important changes in protein properties for only three of them; lysines 72,73 and 79. Optical spectroscopy, electron and nuclear paramagnetic resonance, electron spin echo envelope modulation, and molecular weight studies, as well as the unique features of their reaction with cytochrome-c oxidase, indicate that in the oxidized state the modification of these lysines resulted in equilibria between two different states of iron ligation: the native state, in which the metal is coordinated by the methionine-80 sulfur, and a new state in which this ligand is displaced by the sulfhydryl groups of the elongated side chains. The reduction potentials of the TP Lys-72 and the TP Lys-79 derivatives were 201 and 196 millivolt, respectively, indicating that the equilibria favored the sulfhydryl ligated state by 1.5 and 1.7 kcal/mol, respectively. In the ferric state, the protein modified at lysine 72 remained stable as a monomer, but that modified at lysine 73 dimerized rapidly through disulfide bond formation, while the TP Lys-79 cytochrome c dimerized with a half-time of approx. 3 h, both recovering the native-like iron ligation. By contrast, in the ferrous state the monomeric state and the native ligation were preserved in all cases, indicating that the affinity of the cytochrome-c ferrous iron for the methionine-80 sulfur is particularly strong. The dimerized derivatives lost most, but not all, of the capability of the native protein for electron transfer from ascorbate-TMPD to cytochrome-c oxidase.

Animals↗
PubMed1987

Differences in the association of the progesterone receptor ligated by antiprogestin RU38486 or progestin ORG 2058 to chromatin components.

We assessed the hypothesis that due to variations in the conformation of the progesterone receptor induced by the antiprogestin RU38486 compared to the progestin ORG 2058, differences may result in the association of the receptor with some of the chromatin components. The physical properties of the receptor-bound chromatin fragments released by micrococcal nuclease digestion were characterized by sucrose gradient sedimentation and by gel filtration on Agarose A-1.5m or Agarose A-5m columns. The nuclear fraction was isolated from T47D cells previously exposed to 0.1 microM [3H]RU38486 or 0.1 microM [3H]ORG 2058. Micrococcal nuclease digestion solubilized two receptor forms sedimenting at 4.4 S and 6.3 S for the antiprogestin bound receptor and only one receptor at 4.4 S for the progestin ligated receptor. High-salt buffer dissociated either the antiprogestin or the progestin-bound receptor to smaller receptor forms sedimenting at 3.5 S. Chemical cross-linking with the cross-linker 2-iminothiolane of the micrococcal nuclease solubilized receptor forms resulted in 6.7-S and 4.4-S forms sedimenting on 0.4 M KCl gradients for the antiprogestin and progestin ligated receptors, respectively. Stokes radii of 7.3 nm and 6.4 nm were determined by gel filtration in 0.4 M KCl for the 6.7-S and the 4.4-S receptor forms, respectively. Using the sedimentation coefficient and the Stokes radius, molecular weights of 202,000 and 116,000 were calculated for the antiprogestin and progestin ligated receptors. We conclude that the micrococcal nuclease solubilized antiprogestin ligated receptor is associated with additional or different chromatin components compared to the progestin bound receptor.

Cell Line↗
PubMed1990

Systemic hypotension and pressor responsiveness in cholestasis. A study in conscious 3-day bile duct ligated rats.

It has been postulated that the physiological basis for systemic hypotension in cholestatic liver disease is the attenuated responsiveness of the cardiovascular system to sympathetic stimulation. Using conscious 3-day bile duct ligated rats, we tested this hypothesis by measuring the vasopressor and vasodilator responses following intravenous infusions of norepinephrine, tyramine, angiotensin II, angiotensin I and isoproterenol, in conjunction with the pressor responses to a head-up vertical tilt and a controlled hemorrhage. The results were compared to those obtained in conscious sham-operated rats. Bile duct ligation reduced the mean arterial blood pressure without a significant increase in heart rate. The pressor responses to the aforementioned drugs obtained in the bile-duct ligated rats were significantly attenuated from those the sham-operated rats. In contrast, bile duct ligation had no effect on the pressor responses to tilting and hemorrhage when compared to the responses obtained in the sham-operated rats. Despite the presence of systemic hypotension and attenuation of pressor response to vasoactive drugs, the ability of the cardiovascular system to respond to physiological stimuli appears to be intact in this model. Therefore, we conclude that blunted pressor responsiveness of the cardiovascular system is probably not an important physiological determinant of systemic hypotension in cholestatic liver disease.

Animals↗
PubMed1994

Partial sciatic nerve ligation results in an enlargement of the receptive field and enhancement of the response of dorsal horn neurons to noxious stimulation by an adenosine agonist.

In this study we examined the effect of partial sciatic nerve ligation (PSNL) on the receptive field size, the baseline firing rate (BFR) and the response of spinal dorsal horn (DH) neurons to mechanical stimulation. In addition, we tested the effect of adenosine agonist, 5'-N-ethylcarboxamide-adenosine (NECA), and the adenosine antagonist caffeine on these parameters. Adult male Sprague-Dawley animals were used. One-third to one-half of the right sciatic nerve was tightly ligated. Unanesthetized animals were tested for their response to mechanical stimulation using Von Frey filaments and a blunt probe. The mean force that produced a paw withdrawal response in the operated animals was significantly less than the force that produced withdrawal in unoperated animals (median: 103.5 vs. 259.7; P < 0.001 for the paw ipsilateral to the ligation). Extracellular recordings were made from nociceptive-specific DH neurons located in laminal I-V of chloral hydrate-anesthetized rats. Recordings were made from 38 neurons in the right and 29 cells in the left DH of unoperated and 40 cells in right and 41 cell in the left DH of operated animals. The BFRs of neurons recorded in the operated animals were not significantly different from those recorded in normal animals. The mean receptive field size (RFS) of neurons (both ipsilateral and contralateral to the ligation) in the operated animals was significantly larger than the RFS of unoperated animals (right side: 180 +/- 2.8 mm2 compared to 66 +/- 2.3 mm2; left side: 93 +/- 31 compared to 65 +/- 21). Twenty-four percent of all neurons in the operated group had bilateral receptive fields; in contrast, only 3% of the neurons in the control animals showed bilateral receptive fields. To examine the effects of adenosine agonist and antagonist, NECA and caffeine were applied next to the recording electrode.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine↗
PubMedDate not supplied

Induction of antibacterial activity in the haemolymph of the silkworm, Bombyx mori, by injection of formalin-treated Escherichia coli K-12 in the anterior and posterior body part of the ligated larvae.

1. Induction of antibacterial activity was investigated in the ligated fifth instar larvae of the silkworm, Bombyx mori, by injection of formalin-treated Escherichia coli K-12 into the haemocoel in the anterior and in the posterior body part, followed by activity determination by inhibition zone assay of the haemolymph at 12 and 24 hr after immunization. 2. At 12 hr after immunization, high antibacterial activity, approximately 6.8-7.5 mm in the anterior body part and 4.5-6.4 mm in the posterior body part in diameter of a clear zone (2.0 mm for no activity) was detectable in day 3 larvae. This result was in good contrast to expression of lectin gene in the ligated flesh fly, Sarcophaga peregrena (Shiraishi and Natori, 1988, FEBS Lett. 232, 163-166), in which only the anterior part of insect responded to stimulus of injury. 3. Antibacterial activity at 24 hr after immunization in days 3 or 4 ligated larvae was lower than that at 12 hr; 4.0-4.5 mm of activity was observed in the anterior body part, and no activity was observed in day 3 ligated larvae in the posterior body part. 4. Acid polyacrylamide gel electrophoresis of the haemolymph of immunized insects followed by overlay assay showed that the size of antibacterial activity bands were similar between the haemolymph from 12 hr and from 24 hr, and between the anterior and the posterior body part. This result was contradictory to the observation of activity by inhibition zone assay. The activity bands were associated with peptides that were similar to cecropin-like peptides A and B in the silkworm.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2005

Structure-independent and quantitative ligation of single-stranded DNA.

Ligation of an adapter oligonucleotide to a single-stranded cDNA is central to many molecular biology techniques. Current single-stranded ligation approaches suffer from low efficiencies and are strongly inhibited by preexisting DNA secondary structure. We develop an approach for ligating low concentrations of single-stranded DNAs to a DNA adapter with near-quantitative efficiency, unaffected by secondary structure in the target DNA. This efficient DNA ligation reaction will facilitate development of robust procedures for quantifying small amounts of highly structured cDNAs and their RNA templates.

Base Sequence↗
PubMed2006

Biphasic activation of liver regeneration-associated signals in an early stage after portal vein branch ligation.

At an early stage in liver regeneration, a variety of factors including transcriptional factors, proinflammatory cytokines, and proto-oncogenes are activated or expressed. However, these responses are affected by surgical stress in the conventional portal vein branch ligation model (PVL). We sought to determine the net activation of early regeneration-associated signals after PVL using a newly developed non-surgical stress PVL model. Male Wistar rats were assigned into two groups, a stress-PVL (sPVL) model with laparotomy and portal vein branch ligation, and a non-stress-PVL (nsPVL) one subjected to portal vein branch ligation 1 week after laparotomy in which the effects of surgical stress were subsided. The activation of transcriptional factors and expression of immediate early genes were analyzed at an early time point (within 24 h) by Western blotting and RT-PCR, respectively. A monophasic upregulation of nuclear factor-kappaB (NF-kappaB) and phosphorylated-signal transducer and activator of transcription 3 (p-STAT3) peaked at 3h after sPVL was observed. In contrast, the expression pattern of these factors was biphasic (first peak at 0.5-1 h, second peak at 3-6 h) in nsPVL group. The expression pattern of immediate early genes showed a similar trend between sPVL and nsPVL. cDNA array analysis for the non-ligated lobe at 2 h after PVL revealed a much higher gene activation in sPVL than in nsPVL. These results indicate that previously observed activation pattern of regeneration-associated signals after PVL is significantly affected by the effect of laparotomy and our results using nsPVL model may more accurately represent liver regeneration-associated signal pattern.

Animals↗
PubMed2006

Ligation of centrocyte/centroblast marker 1 on Epstein-Barr virus--transformed B lymphocytes induces cell death in a reactive oxygen species--dependent manner.

After primary infection of B cells with Epstein-Barr virus (EBV), infected B cells express several viral homologs of human genes that promote activation (LMP1 and CD40) or survival (BHRF and BCL2). EBV-infected B cells also express germinal center phenotype markers, such as CD77, PNA, CD95, and CD38. This transformation of B cells by EBV infection resembles normal B-cell activation and differentiation arising in the germinal center. In the present study, we found that EBV-transformed B cells expressed centrocyte/centroblast marker 1 (CM1), a possible marker of GC B cells and an inducer of their apoptosis. Moreover, ligation of CM1 on EBV-transformed B cells by immobilized anti-CM1 monoclonal antibody induced cell death. The ligation of CM1 immediately increased the generation of intracellular reactive oxygen species (ROS) and disrupted the mitochondrial membrane potential. Pretreatment with N-acetyl cystein (an ROS inhibitor) almost completely blocked this cell death, but Z-VAD-fmk (a caspase inhibitor) did not. We further investigated whether apoptosis-inducing factor (AIF) and endonuclease G (EndoG), which are both related to caspase-independent cell death, would be translocated to the nucleus during the ligation of CM1. We found that AIF and EndoG were released to the cytosplam but not translocated to the nucleus. Moreover, cytochalasin D, a cytoskeleton disruptor, rescued the cells from CM1-mediated cell death and blocked ROS generation. Therefore, it is conceivable that CM1 signaling might provoke cytoskeleton polymerization and trigger ROS generation. Taking these observations together, we conclude that the ligation of CM1 on EBV-transformed B cells can cause cell death via the ROS produced by F-actin polymerization in a caspase-independent manner, although this cell death might be unrelated to AIF and EndoG release from the mitochondria.

Acetylcysteine↗
PubMed2004

Ligation of RARgamma inhibits proliferation of phytohaemagglutinin-stimulated T-cells via down-regulating JAK3 protein levels.

The mechanisms whereby Vitamin A regulates the immune system are poorly understood. We have shown previously that retinoic acids, the Vitamin A derivatives, promote both apoptosis of neglected thymocytes and the activation-induced cell death of peripheral T-cells via ligating the nuclear retinoid receptor (RAR) gamma. In the present study, we found that human peripheral T-cells express RARalpha and gamma, but not RARbeta. Increasing concentrations of 9-cis RA inhibited phytohaemagglutinin (PHA)-induced proliferation of T-cells, an effect that could be mimicked only by addition of RARgamma agonists and could be inhibited by an RARgamma antagonist. Interleukin-2 (IL-2) produced is known to mediate PHA-induced proliferation of T lymphocytes. Ligation of RARgamma did not affect the PHA-induced high affinity IL-2 receptor expression, slightly reduced the PHA-induced IL-2 production, but interfered with the IL-2-mediated signal transduction resulting in inhibition of PHA-induced phosphorylation of retinoblastoma protein and of up-regulation of Bcl-2. Janus kinases JAK1 and JAK3 play a determinant role in IL-2-dependent signal transduction. Ligation of RARgamma did not affect the levels of JAK1, but prevented IL-2-induced expression of JAK3 resulting in inhibition of PHA-induced phosphorylation of Stat5 molecules. Our data suggest that the previously observed toxic effect of high concentrations of retinoids on the immune system might be mediated via formation of 9-cis RA, which via ligation of RARgamma not only induces cell death in immature thymocytes, but inhibits proliferation of T-cells as well.

Alitretinoin↗
PubMed2004

The efficiency of mispaired ligations by lambda integrase is extremely sensitive to context.

The integrase protein (Int) of phage lambda is a well-studied representative of the tyrosine recombinase family, whose defining features are two sequential pairs of DNA cleavage/ligation reactions that proceed via a 3' phosphotyrosine covalent intermediate to first form and then resolve a Holliday junction recombination intermediate. We devised an assay that takes advantage of DNA hairpin formation at one Int target site to trap Int cleavages at a different target site, and thereby reveal iterative cycles of cleavage and ligation that would otherwise be undetected. Using this assay and others to compare wild-type Int and a mutant (R169D) defective in forming proper dimer/tetramer interfaces, we found that the efficiency of "bottom-strand" DNA cleavage by wild-type Int, but not R169D, is very sensitive to the base-pair at the "top-strand" cleavage site, seven base-pairs away. We show that this is related to the finding that hairpin formation involving ligation of a mispaired base is much faster for R169D than for wild-type Int, but only in the context of a multimeric complex. During resolution of Holliday junction recombination intermediates, wild-type Int, but not R169D, is very sensitive to homology at the sites of ligation. A long-sought insight from these results is that during Holliday junction resolution the tetrameric Int complex remains intact until after ligation of the product helices has been completed. This contrasts with models in which the second pair of DNA cleavages is a trigger for dissolution of the recombination complex.

Bacteriophage lambda↗
PubMed2004

Development of a versatile cassette for directional genome walking using cassette ligation-mediated PCR and its application in the cloning of complete lipolytic genes from Bacillus species.

Since the invention of the PCR technology, adaptation techniques to clone DNA fragments flanking the known sequence continue to be developed. We describe a perfectly annealed cassette available in almost unlimited quantities with variable sticky-and blunt-end restriction enzyme recognition sites for efficient restriction and ligation with the restricted target genomic DNA. The cassette provides a 200-bp sequence, which is used to design a variety of cassette-specific primers. The dephosphorylation prevents cassette self-ligation and creates a nick at the cassette: target genome DNA ligation site suppressing unspecific PCR amplifications. We introduce the single-strand amplification PCR (SSA-PCR) technique where a lone known locus-specific primer is firstly used to enrich the targeted template DNA strand resulting in significant PCR product specificity during the second round conventional nested PCR. The distance between the known locus-specific primer and the nearest location of the restriction enzyme used determined the length of the obtained PCR product. We used this technique to walk downstream into the isochorismatase and upstream into the hypothetical conserved genes flanking the mature extracellular lipase gene from Bacillus licheniformis. We further demonstrated the potential of the technique as a cost-effective method during PCR-based prospecting for novel genes by designing "universal" degenerate primers that detected homologues of Family VII bacterial lipolytic genes in Bacillus species. The cassette ligation-mediated PCR was used to clone complete nucleotide sequences encoding functional lipolytic genes from B. licheniformis and Bacillus pumilus.

Amino Acid Sequence↗