Elucidation of the mechanism of CryIIIA overproduction in a mutagenized strain of Bacillus thuringiensis var. tenebrionis

NB176 is a Bacillus thuringiensis mutant derived by λ-irradiation of NB125 Bacillus thuringiensis var. tenebrionis (Krieg). It exhibits two interesting phenotypes: (i) oligosporogeny and (ii) twofold to threefold overproduction of the CryIIIA protein. Southern profiles of the NB176 strain showed an additional copy(s) of the cryIIIA gene located on a 4 kb HindIII fragment, in addition to the expected cryIIIA gene on a 3 kb HindIII fragment. Each cryIIIA gene-bearing HindIII fragment was cloned from NB176. The restriction map of the 3 kb HindIII fragment was identical to that published by Donovan and coworkers. Sequencing of the 4 kb HindIII fragment showed no alterations in the promoter region of the cryIIIA gene but did show replacement of the region immediately following the cryIIIA open reading frame with a sequence encoding a transposase with 50% amino acid homology to that of Tn 1000. These findings suggest that the overproduction phenotype of NB176 results from extra copies of the cryIIIA gene produced from a transposition event(s) induced or stabilized by γ-irradiation. Integration of additional copies of the cryIIIA gene into the native 90MDa plasmid of the wild-type B. thuringiensis var. tenebrionis strain resulted in strains that made enormous crystals, many possessing greatly enhanced insecticidal activity.     

Receptor interactions of the position 4 side chains of angiotensin II analogues: Importance of aromatic ring quadrupole

A triad of interacting group (TyrOH? His$ \underline\ominus$O₂C) in angiotensin II (ANG II) has been postulated to create the tyrosinate anion pharmacophore (tyanophore) responsible for receptor activation/triggering (Biochim. Biophys. Acta 1991, 1065, 21). In the present study we investigated the effects on bioactivity of substituting the Tyr⁴ residue in [Sar¹]ANG II with other anionic or electronegative amino acids, and with a number of aromatic amino acids lacking a hydroxyl group. [Sar¹ Nva(δ-OH)⁴]ANG II, [Sar¹ Nva(δ-OCH₃)⁴]ANG II, [Sar¹ Met⁴]ANG II, [Sar¹ Gln⁴]ANG II, [Sar¹ Glu⁴]ANG II and [Sar¹ DL -Alg⁴]ANG II had agonist activities in the rat isolated uterus assay of 4, 3, 19, 10, > 0.1 and > 0.1%, respectively, of that of ANG II. [Sar¹ Nal⁴]ANG II, [Sar¹ Pal⁴]ANG II, [Sar¹ DL -Phg(4′-F)⁴]ANG II, [Sar¹ Phe(4′-F)⁴]ANG II, [Sar¹ Phe(F₅)⁴]ANG II and [Sar¹ His⁴]ANG II had agonist activities of 4.5, 7, < 0.1, 0.2, 1 and 0.6%, respectively. All peptides investigated were devoid of measurable antagonist activity except [Sar¹] Phe(4′-F)⁴ ANG II (pA₂ = 7.7). These findings illustrate that anionic or electronegative aliphatic side chains replacing tyrosinate at position 4 can partially activate the angiotension receptor. For ANG II analogues containing an aromatic amino acid other than Tyr at position 4, ligand binding and agonist activity are not dependent on the electronegativity or dipole moment of the aromatic ring, or on the ability of the 4′ ring substituent to accept a proton. Modelling based on ab initio calculations of aromatic ring multipoles illustrate that the apparent binding affinity (PA₂) of ANG II analogues is associated with a perpendicular electrostatic interaction of the position 4 aromatic ring with a receptor-based group. In addition, intramolecular interactions providing for the conformation of the ligand as it approaches its receptor appear to have a role in determining agonist vs antagonist activity.     

Apparent regression of the CGG repeat in FMR1 to an allele of normal size

The fragile X syndrome is the result of amplification of a CGG trinucleotide repeat in the FMR1 gene and anticipation in this disease is caused by an intergenerational expansion of this repeat. Although regression of a CGG repeat in the premutation range is not uncommon, regression from a full premutation (>200 repeats) or premutation range (50–200 repeats) to a repeat of normal size (<50 repeats) has not yet been documented. We present here a family in which the number of repeats apparently regressed from approximately 110 in the mother to 44 in her daughter. Although the CGG repeat of the daughter is in the normal range, she is a carrier of the fragile X mutation based upon the segregation pattern of Xq27 markers flanking FMR1. It is unclear, however, whether this allele of 44 repeats will be stably transmitted, as the daughter has as yet no progeny. Nevertheless, the size range between normal alleles and premutation alleles overlap, a factor that complicates genetic counseling.     

Thermodynamics of ubiquitin unfolding

The energetics of ubiquitin unfolding have been studied using differential scanning microcalorimetry. For the first time it has been shown directly that the enthalpy of protein unfolding is a nonlinear function of temperature. Thermodynamic parameters of ubiquitin unfolding were correlated with the structure of the protein. The enthalpy of hydrogen bonding in ubiquitin was calculated and compared to that obtained for other proteins. It appears that the energy of hydrogen bonding correlates with the average length of the hydrogen bond in a given protein structure. © 1994 John Wiley & Sons, Inc.     

Localization of a New Type of X-Linked Liver Glycogenosis to the Chromosomal Region Xp22 Containing the Liver α-Subunit of Phosphorylase Kinase (PHKA2)

We describe here a new type of X-linked liver glycogen storage disease. The main symptoms include liver enlargement and growth retardation. The clinical and biochemical abnormalities of this glycogenosis are similar to those of classical X-linked liver glycogenosis due to phosphorylase kinase deficiency (XLG). However, in contrast to patients with XLG, the patients described here have no reduced phosphorylase kinase activity in erythrocytes and leukocytes, and no enzyme deficiency could be found. Linkage analysis of four families with this X-linked type of liver glycogenosis assigned the disease gene to Xp22. Lod scores obtained with the markers DXS987, DXS207, and DXS999 were 3.97, 2.71, and 2.40, respectively, all at 0% recombination. Multipoint linkage analysis localized the disease gene between DXS143 and DXS989 with a maximum lod score of 4.70 at θ = 0, relative to DXS987. As both the classical XLG gene and the liver α-subunit of PHK (PHKA2) are also located in Xp22, this variant type of XLG may be allelic to classical XLG, and both diseases may be caused by mutations in PHKA2. Therefore, we propose to classify XLG as XLG type I (the classical type of XLG) and XLG type II (the variant type of XLG).     

Natal philopatry in passerine birds: genetic or ecological influences?

The degree of natal philopatry (the likelihood that individualsbreed at or near their place of origin) can influence the extentof inbreeding in animal populations. Passerine birds have beencited as typically showing high natal philopatry, and natalphilopatry has been proposed as an adaptation to promote optimalinbreeding. A review of published and unpublished studies ofpasserines showed that natal philopatry was typically low, somaintaining a high level of inbreeding appears relatively unimportantfor such birds. Rather, natal philopatry appeared to be morestrongly influenced by ecological factors. Migratory passerineexhibited low natal philopatry compared to resident passerines,as predicted if dispersal costs for young birds are an importantdeterminant of natal philopatry. The erroneous view that natalphilopatry for passerines is generally high has resulted froma reporting bias toward resident species that have sufficientnatal philopatry to study. Natal philopatry was found to beevolutionarily labile; populations of the same species and pairsof closely related species that differed in their degree ofisolation differed considerably in their degree of philopatry.Future studies of natal philopatry should consider both theecological factors that could affect dispersal costs and thereporting biases that influence which data on philopatry tendto be reported.     

Detection of Proteins in Normal and Alzheimer's Disease Cerebrospinal Fluid with a Sensitive Sandwich Enzyme-Linked Immunosorbent Assay

Abstract— Alzheimer's disease is a progressive degenerative dementia characterized by the abundant presence of neurofibrillary tangles in neurons. This study was designed to test whether the microtubule-associated protein, a major component of neurofibrillary tangles, could be detected in CSF. Additionally, we investigated whether CSF levels were abnormal in Alzheimer's disease as compared with a large group of control patients. We developed a sensitive sandwich enzyme-linked immunosorbent assay using AT120, a monoclonal antibody directed to human, as a capturing antibody. With this technique, the detection limit for was less than 5 pg/ml of CSF. Using ATS, which recognizes abnormally phosphorylated ser-ines 199–202 in, the detection limit was below 20 pg/ml of CSF. However, with AT8, we found no immunoreactiv-ity in CSF, suggesting that only a small fraction of CSF contains the abnormally phosphorylated AT8 epitope. Our results indicate that CSF levels are significantly increased in Alzheimer's disease. Also, CSF levels in a large group of patients with a diversity of neurological diseases showed overlap with CSF levels in Alzheimer's disease.     

PEAK SHIFTS AND POLYMORPHISM DURING PHASE THREE OF WRIGHT'S SHIFTING-BALANCE PROCESS

The third phase of Wright's shifting-balance theory involves the export of adaptive gene combinations from one subpopulation to another. Previous results have demonstrated that this can occur at very low migration rates, but it has been argued that this simply reflects the ability of migration to overcome selection and fix any (even deleterious) alleles. Here, previous analyses are extended by concentrating on the critical balance between forward and reverse migration rates that still allows phase III to proceed. It is shown that selective advantage, dominance, recombination rate, and the number of loci all affect the ability of a genotype to invade and become fixed in a new subpopulation, but it is unlikely that phase III will occur in the absence of differential migration unless the invading genotype consists of a few dominant loci with a large selection advantage, spreading into a few populations of lower fitness. Therefore, as was envisioned by Wright, differential migration from more to less fit populations will be necessary for phase III to occur under most circumstances.     

Founder effect in a Belgian-Dutch fragile X population

For many years, the high prevalence of the fragile X syndrome was thought to be caused by a high mutation frequency. The recent isolation of the FMR1 gene and identification of the most prevalent mutation enable a more precise study of the fragile X mutation. As the vast majority of fragile X patients show amplification of an unstable trinucleotide repeat, DNA studies can now trace back the origin of the fragile X mutation. To date, de novo mutations leading to amplification of the CGG repeat have not yet been detected. Recently, linkage disequilibrium was found in the Australian and US populations between the fragile X mutation and adjacent polymorphic markers, suggesting a founder effect of the fragile X mutation. We present here a molecular study of Belgian and Dutch fragile X families. No de novo mutations could be found in 54 of these families. Moreover, we found significant (P < 0.0001) linkage disequilibrium in 68 unrelated fragile X patients between the fragile X mutation and an adjacent polymorphic microsatellite at DXS548. This suggests that a founder effect of the fragile X mutation also exists in the Belgian and Dutch populations. Both the absence of new mutations and the presence of linkage disequilibrium suggest that a few ancestral mutations are responsible for most of the patients with fragile X syndrome.     

Reactions Catalyzed by Peg-Modified α-Chymotrypsin in Organic Solvents. Influence of Water Content and Degree of Modification

-Chymotrypsin was modified with cyanuric chloride activated monomethoxypolyethylene glycol (MPEG) with molecular weights 1900 and 5000. Using the higher molecular weight MPEG a product that was soluble in benzene at moderate levels of modification was obtained, whereas with MPEG 1900 almost all the enzyme's amino groups had to be modified for dissolving the conjugate. The catalytic activity decreased with increasing degree of substitution. Apparent V_max was considerably higher for the less modified enzyme preparation than for the more modified one, while K_m,app stayed almost constant. The modified enzyme was used for peptide synthesis. The reaction was dependent on the content of dissolved water. Both V_max,app and K_m,app increased with increasing water content. It was possible to achieve a process with complete conversion of substrate to dipeptide.