An effective alternate cloning strategy for unstable mouse genomic sequences

Unstable mammalian genomic sequences frequently underwent spontaneous rearrangement during the bacterial cloning process. When the flanking sequences of an INSM1 gene comprised of 3.0 and 4.5 kb were subcloned into a targeting vector for a gene deletion study, both the genomic sequences underwent spontaneous rearrangement. Neither the usage of recombinase-free Escherichia coli competent cells nor lowering the culture incubation temperature averted the recombination events. Co-transformation of a methyltransferase vector, pAIT2, with the targeting vector had little effect in preventing recombination through methylation of the plasmid DNA. Here, we show that a single-copy cloning technique is effective to clone the unstable mouse genomic DNA into the targeting vector.     

An alternate diafiltration strategy to mitigate protein precipitation for low solubility proteins

Application of the minimum diafiltration (DF) time solution for a monoclonal antibody resulted in a 20‐h process time rather than the expected 12 h. Further investigation indicated high turbidity associated with a product solubility issue that caused a flux decline. As a result, the gel flux model and the associated minimum DF time were not predictive. Multiwell plate solubility screening confirmed that the protein passed through a region of low solubility during the ultrafiltration step. Multiple approaches to address this issue were considered and a new strategy involving variable volume diafiltration (VVDF) was developed. Process modeling and simulation were used to predict performance and to select a value of the DF ratio control parameter (buffer flow/permeate flow = 0.65). Feasibility testing at the bench and pilot scales confirmed that the new strategy reduced solubility issues, fit within existing manufacturing tank volume and system area constraints, matched model predictions, and did not present significant implementation issues. Recommendations are made regarding the general value of this strategy, when it should be used, and how to implement it. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:646–655, 2014     

An adaptive, object oriented strategy for base calling in DNA sequence analysis.

An algorithm has been developed for the determination of nucleotide sequence from data produced in fluorescence-based automated DNA sequencing instruments employing the four-color strategy. This algorithm takes advantage of object oriented programming techniques for modularity and extensibility. The algorithm is adaptive in that data sets from a wide variety of instruments and sequencing conditions can be used with good results. Confidence values are provided on the base calls as an estimate of accuracy. The algorithm iteratively employs confidence determinations from several different modules, each of which examines a different feature of the data for accurate peak identification. Modules within this system can be added or removed for increased performance or for application to a different task. In comparisons with commercial software, the algorithm performed well.     

An alternate method for synthesis of double-stranded DNA segments

Recent progress in the chemical synthesis of DNA has now made it possible to rapidly synthesize single-stranded DNAs over 40 bases in length. We have taken advantage of these longer DNAs in assembling and cloning a 132-base pair gene segment coding for amino acids 126 through the stop codon of human leukocyte interferon alpha 2. The method used involves DNA polymerase I-mediated repair synthesis of synthetic oligonucleotide substrates having short stretches of complementary sequence at their 3' termini. In the presence of DNA polymerase I and the four deoxyribonucleoside triphosphates, those primer-templates are converted to full length double-stranded DNAs. The economy in chemical synthesis using this approach is substantial with a greater than 40% reduction in the amount of chemical synthesis required as compared with the conventional approach. We describe in detail this methodology for the biochemical assembly of long gene segments from synthetic oligodeoxyribonucleotides.     

An improved strategy for generating a family of unidirectional deletions on large DNA fragments

A modification of the Barnes "kilo-sequencing" method is described. The procedure presented here makes it possible to obtain a series of nested deletions on large DNA fragments in only two days. It applies to double-stranded DNA, and thus can be used with plasmids as well as the M13mp series of bacteriophages. The main improvements are the use of a second restriction enzyme, which makes it possible to begin the deletions at any site on the DNA fragment, and the use of mung bean nuclease for trimming the DNA edges so that any restriction enzyme can be used. This method, using a pUC vector and sequencing on double-stranded DNA, would make it possible to read a DNA nucleotide sequence on both strands starting with only one construction.     

An alternate method of descenting skunks

Striped skunk (Mephitis mephitis) scent glands were ligated closed with waxed dental floss to allow them to be handled during toxicological studies without fear of scenting. This descenting technique was more rapid and less traumatic than scent gland removal. Thirty-four skunks were kept for less than or equal to 127 days and did not display behavioral or physical abnormalities due to this procedure.     

An alternate high yielding purification method for Clitoria ternatea lectin

In our previous publication we had reported the purification and characterization of Clitoria ternatea agglutinin from its seeds on fetuin CL agarose affinity column, designated CTA [A. Naeem, S. Haque, R.H. Khan. Protein J., 2007]. Since CTA binds beta-d-galactosides, this lectin can be used as valuable tool for glycobiology studies in biomedical and cancer research. So an attempt was made for a high yielding alternative purification method employing the use of asialofetuin CL agarose column for the above-mentioned lectin, designated CTL. The fetuin affinity purified agglutinin was found similar to asialofetuin affinity purified lectin in SDS pattern, HPLC and N-terminal sequence. The content of lectin was found to be 30mg/30g dry weight of pulse. The yield was 2.8% as compared to 0.3% obtained on fetuin column. The number of tryptophan and tyrosine estimated was four and six per subunit.     

An alternate targeting pathway for procathepsin L in mouse fibroblasts

In transformed mouse fibroblasts, a significant proportion of the lysosomal cysteine protease cathepsin L remains in cells as an inactive precursor which associates with membranes by a mannose phosphate-independent interaction. When microsomes prepared from these cells were resolved on sucrose gradients, this procathepsin L was localized in dense vesicles distinct from those enriched for growth hormone, which is secreted constitutively when expressed in fibroblasts. Ultrastructural studies using antibodies directed against the propeptide to avoid detection of the mature enzyme in lysosomes revealed that the proenzyme was concentrated in dense cores within small vesicles and multivesicular endosomes which labeled with antibodies specific for CD63. Consistent with the resemblance of these cores to those of regulated secretory granules, secretion of procathepsin L from fibroblasts was modestly stimulated by phorbol, 12-myristate, 13-acetate. When protein synthesis was blocked with cycloheximide and lysosomal proteolysis inhibited with leupeptin, procathepsin L was found to gradually convert to the active single-chain protease. The data suggest that when synthesis levels are high, a portion of the procathepsin L is packaged in dense cores within multivesicular endosomes localized near the plasma membrane. Gradual activation of this proenzyme achieves targeting of the proenzyme to lysosomes by a mannose phosphate receptor-independent pathway.     

An alternate and reversible method for flight restraint of cranes

Flight restraint is important for zoos, safaris, and breeding centers for large birds. Currently used techniques for flight restraint include both surgical and non-surgical approaches. Surgical approaches usually cause permanent change to or removal of tendon, patagial membrane, or wing bones, and can cause pain and inflammation. Non-surgical approaches such as clipping or trimming feathers often alter the bird's appearance, and can damage growing blood feathers in fledglings or cause joint stiffness. We observed microstructure of primary feathers of the red-crowned crane (Grus japonensis) and found that the width of barbs is a determinative factor influencing vane stiffness and geometric parameters. We hypothesized that partial longitudinal excision of barbs on the ventral surface of the primary feathers would reduce the stiffness of the vane and render the feathers unable to support the crane's body weight during flight. Furthermore, we hypothesized that this modification of barbs would also change the aerodynamic performance of feathers such that they could not generate sufficient lift and thrust during flapping to enable the bird to fly. We tested this hypothesis on a red-crowned crane that had normal flight capability by excising the ventral margin of barbs on all 10 primaries on the left wing. The bird was unable to take off until the modified feathers were replaced by new ones. Removal of barbs proved to be a simple, non-invasive, low-cost and reversible method for flight restraint. It is potentially applicable to other large birds with similar structural characteristics of primary feathers.     

An alternate pathway for the processing of the prolipoprotein signal peptide in Escherichia coli

Previous studies showed that when the signal sequence plus 9 amino acid residues from the amino terminus of the major lipoprotein of Escherichia coli was fused to beta-lactamase, the resulting hybrid protein was modified, proteolytically processed, and assembled into the outer membrane as was the wild-type lipoprotein (Ghrayeb, J., and Inouye, M. (1983) J. Biol. Chem. 259, 463-467). We have constructed several hybrid proteins with mutations at the cleavage site of the prolipoprotein signal peptide. These mutations are known to block the lipid modification of the lipoprotein at the cysteine residue, resulting in the accumulation of unprocessed, unmodified prolipoprotein in the outer membrane. The mutations blocked the lipid modification of the hybrid protein. However, in contrast to the mutant lipoproteins, the cleavage of the signal peptides for the mutant hybrid proteins did occur, although less efficiently than the unaltered prolipo-beta-lactamase. The mutant prolipo-beta-lactamase proteins were cleaved at a site 5 amino acid residues downstream of the prolipoprotein signal peptide cleavage site. This new cleavage between alanine and lysine residues was resistant to globomycin, a specific inhibitor for signal peptidase II. This indicates that signal peptidase II, the signal peptidase which cleaves the unaltered prolipo-beta-lactamase, is not responsible for the new cleavage. The results demonstrate that the cleavage of the signal peptide is a flexible process that can occur by an alternative pathway when the normal processing pathway is blocked.     

An alternate respiratory pathway in Candida albicans

Usual concentrations of antimycin A, rotenone and EDTA, individally or in combination, reduced aerobic growth rate and cell yield of Candida albicans to about half its normal level and to about the levels of previously-described acetate-negative, cytochrome-complete and aa₃-deficient variants which were little affected by the inhibitors. Anaerobic conditions (not affected by antimycin A) reduced growth rate and cell yield of all cultures-including that of a nonrespiring aa₃, b-deficient mutant-to low, equal levels. Antimycin A but not rotenone prevented growth of the normal strain on ethanol medium. Cyanide and antimycin A blocked most of the respiration of the normal strain and cytochrome-complete variant, but did not affect that of the cytochrome aa₃-deficient mutant. Rotenone and EDTA did not affect respiration of any of the cultures. SHAM blocked cyanide- and antimycin A-insensitive respiration and prolonged the lag phases of the three respiring cultures, especially in the presence of antimycin A, but alone increased oxygen-uptake rate of the cytochromecomplete cultures while curtailing that of the cytochrome aa₃-deficient mutant. Resting cells, especially wild-type, grown in medium containing antimycin A exhibited lowered oxygen-uptake rate, which was increased upon the addition of cyanide or antimycin A. Antimycin A stimulated, but cyanide inhibited, respiration of cytochrome-complete cultures grown in the presence of rotenone but did not affect that of the cytochrome aa₃-deficient mutant. SHAM inhibited respiration of all antimycin A- or rotenone-grown cultures. The high rate of respiration of C. albicans in the presence of inhibitors for three sites of electron transport in the conventional oxidative pathway, the inhibition of this respiration by SHAM and its loss by the absence of cytochrome b, indicate an alternate oxidative pathway in this organism which crosses the conventional one at cytochrome b.This work was supported by Public Health Service Graduate Dental Training Grant DE 00144 and the Graduate School and the Department of Microbiology, Southern Illinois University.     

A double base change in alternate base pairs induced by ultraviolet irradiation in a glycine transfer RNA gene

Summary The glyUsu _AGA mutation affects Escherichia coli tRNA _GGG ^{G1 y} , changing it to an AGA missense suppressor tRNA. Sequence studies have shown that the mutation involves a double base substitution at the first and third positions of the tRNA anticodon, the result being a change in the anticodon from CCC to UCU. A system has been developed to facilitate the detection of this novel mutation, and we have shown that ultraviolet irradiation and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) are effective in causing the double base change. A single observation of the mutation occurring spontaneously has been made also. The frequency of MNNG-induced glyUsu _AGA mutations is compatible with their being caused by two separate mutagenic events. The frequency of UV-induced glyUsu _AGA mutations, however, strongly suggests that the occurrence of one base substitution strongly enhances the chance of finding the second substitution at the alternate position.In addition to the double change in the anticodon, the glyUsu _AGA tRNA differs from tRNA _GGG ^{G1 y} in that it bears a modification of the A adjacent to the 3 position of the anticodon. Most likely, this modified base is N-[9-(-D-ribofuranosyl)-purin-6-ylcarbamoyl] threonine.