The m gamma delta-1 element, a small gamma delta (Tn1000) derivative useful for plasmid mutagenesis, allele replacement and DNA sequencing.

Transposon gamma delta (Tn1000), a 6-kb member of the Tn3 family, is widely used for plasmid mutagenesis. A 1.8-kb derivative of gamma delta was constructed that contains the kan gene from Tn5 and the resolution (res) site from gamma delta cloned between 40-bp inverted repeats of gamma delta's delta (delta) end. This element, named m gamma delta-1, lacks the genes encoding transposase and resolvase, and therefore depends on its host to supply transposition and resolution functions. Thus, in strains lacking gamma delta, m gamma delta-1 will not transpose. The m gamma delta-1 element is shown to be useful for mutagenesis of plasmids, DNA sequencing, and allele replacement (in Streptomyces avermitilis).     

The lepidopteran mitochondrial control region: structure and evolution

For several species of lepidoptera, most of the approximately 350-bp mitochondrial control-region sequences were determined. Six of these species are in one genus, Jalmenus; are closely related; and are believed to have undergone recent rapid speciation. Recent speciation was supported by the observation of low interspecific sequence divergence. Thus, no useful phylogeny could be constructed for the genus. Despite a surprising conservation of control-region length, there was little conservation of primary sequences either among the three lepidopteran genera or between lepidoptera and Drosophila. Analysis of secondary structure indicated only one possible feature in common--inferred stem loops with higher-than-random folding energies-- although the positions of the structures in different species were unrelated to regions of primary sequence similarity. We suggest that the conserved, short length of control regions is related to the observed lack of heteroplasmy in lepidopteran mitochondrial genomes. In addition, determination of flanking sequences for one Jalmenus species indicated (i) only weak support for the available model of insect 12S rRNA structure and (ii) that tRNA translocation is a frequent event in the evolution of insect mitochondrial genomes.      

Glutamine repeats and inherited neurodegenerative diseases: molecular aspects

Several dominantly inherited, late onset, neurodegenerative diseases are due to expansion of CAG repeats, leading to expansion of glutamine repeats in the affected proteins. These proteins are of very different sizes and, with one exception, show no sequence homology to known proteins or to each other; their functions are unknown. In some, the glutamine repeat starts near the N-terminus, in another near the middle and in another near the C-terminus, but regardless of these differences, no disease has been observed in individuals with fewer than 37 repeats, and absence of disease has never been found in those with more than 41 repeats. Protein constructs with more than 41 repeats are toxic to E. coli and to CHO cells in culture, and they elicit ataxia in transgenic mice. These observations argue in favour of a distinct change of structure associated with elongation beyond 37–41 glutamine repeats. The review describes experiments designed to find out what these structures might be and how they could influence the properties of the proteins of which they form part. Poly- -glutamines form pleated sheets of β-strands held together by hydrogen bonds between their amides. Incorporation of glutamine repeats into a small protein of known structure made it associate irreversibly into oligomers. That association took place during the folding of the protein molecules and led to their becoming firmly interlocked by either strand- or domain-swapping. Thermodynamic considerations suggest that elongation of glutamine repeats beyond a certain length may lead to a phase change from random coils to hydrogen-bonded hairpins. Possible mechanisms of expansion of CAG repeats are discussed in the light of looped DNA model structures.     

Gamma delta transposase. Purification and analysis of its interaction with a transposon end.

gamma delta, a member of the Tn3 family of prokaryotic transposons, encodes a transposase that binds to the 35-base pair (bp) terminal inverted repeats (IRs) which define the transposing DNA segment. The gamma delta transposase has been overexpressed, identified by molecular weight determination and by immunoblotting, and purified to homogeneity. Production of soluble transposase required the presence of Mg2+ prior to cell lysis. Fractions from a Sephacryl S-300 column contained levels of IR-binding activity that parallel the concentration of transposase, indicating that transposase alone is sufficient for binding to the ends of gamma delta. Hydroxyl radical footprinting indicated that transposase binds to one face of the DNA helix. The protected region extends across the IR and up to 17 bp into the flanking DNA. Integration host factor (IHF), which binds adjacent to transposase, also protects one face of the DNA helix and is shifted about 70 degrees around the helical axis from the transposase protection. Analysis of transposase-DNA complexes by electrophoresis on nondenaturing gels indicated that three complexes, two within the gel and one trapped at the well, result from specific interactions with the IR. The complex in the well and one complex in the gel were analyzed by methylation interference experiments. The results indicate that transposase interacts with specific base pairs between positions 10 and 37 of the IR, a region encompassing three consecutive major and minor grooves. Methylated bases at the very end of the transposon (positions 1-9) and in the flanking DNA did not inhibit transposase binding. Thus, although transposase seems to be in intimate contact throughout the IR of gamma delta and 17 bp of flanking DNA, specific base pair recognition needed for binding appears to be determined by the inner three-quarters of the IR.     

Integration host factor increases the transpositional immunity conferred by gamma delta ends.

The ends of the bacterial transposon gamma delta contain adjacent binding sites for gamma delta transposase and integration host factor (IHF). IHF+ and IHF- strains were used in conjunction with gamma delta transposon ends containing or lacking the site for IHF binding to determine the role that IHF plays in various gamma delta-mediated transposition events. IHF was not essential for the transposition of gamma delta and seemed to decrease its frequency of transposition about threefold. IHF played no role in determining the distribution of gamma delta inserts into a target replicon, nor did it significantly alter the frequency of simple transpositions. The only clear role discerned for IHF and the terminal IHF-binding sites was in transposition immunity. IHF stimulated the immunity of those plasmids that contain an end of gamma delta, provided the end included the terminal IHF-binding site. For both ends, the degree of stimulation of immunity was similar to the stimulation of binding of transposase by IHF.     

Development of cell surface saccharides on embryonic pancreatic cells

Using a battery of seven lectin-ferritin conjugates as probes for cell surface glycoconjugates, we have studied the pattern of plasmalemmal differentiation of cells in the embryonic rat pancreas from day 15 in utero to the early postpartum stage. Our results indicate that differentiation of plasmalemmal glycoconjugates on acinar, endocrine, and centroacinar cells is temporally correlated with development and is unique for each cell type, as indicated by lectin-ferritin binding. Specifically, (a) expression of adult cell surface saccharide phenotype can be detected on presumptive acinar cells as early as 15 d in utero, as indicated by soybean agglutinin binding, and precedes development of intracellular organelles characteristic of mature acinar cells; (b) maturation of the plasmalemma of acinar cells is reached after intracellular cytodifferentiation is completed, as indicated by appearance of Con A and fucoselectin binding sites only at day 19 of development; conversely, maturation of the endocrine cell plasmalemma is accompanied by "loss" (masking) of ricinus communis II agglutinin receptors; and (c) binding sites for fucose lectins and for soybean agglutinin are absent on endocrine and centroacinar cells at all stages examined. We conclude that acinar, centroacinar, and endocrine cells develop from a common progenitor cell(s) whose plasmalemmal carbohydrate composition resembles most closely that of the adult centroacinar cell. Finally, appearance of acinar lumina beginning at approximately 17 d in utero is accompanied by differenetiation of apical and basolateral plasmalemmal domains of epithelial cells, as indicated by enhanced binding of several lectin-ferritin conjugates to the apical plasmalemmal, a pattern that persists from this stage through adult life.     

Isolation and characterization of α‐(1→3)‐glucan‐degrading bacteria from the gut of Diaperis boleti feeding on Laetiporus sulphureus

Bacteria degrading α‐(1→3)‐glucan were sought in the gut of fungivorous insects feeding on fruiting bodies of a polypore fungus Laetiporus sulphureus, which are rich in this polymer. One isolate, from Diaperis boleti, was selected in an enrichment culture in the glucan‐containing medium. The bacterium was identified as Paenibacillus sp. based on the results of the ribosomal DNA analysis. The Paenibacillus showed enzyme activity of 4.97 mU/cm³ and effectively degraded fungal α‐(1→3)‐glucan, releasing nigerooligosaccharides and a trace amount of glucose. This strain is the first reported α‐(1→3)‐glucan‐degrading microorganism in the gut microbiome of insects inhabiting fruiting bodies of polypore fungi.     

B30.2-like domain proteins: update and new insights into a rapidly expanding family of proteins

The B30.2 domain is a conserved region of around 170 amino acids associated with several different protein domains, including the immunoglobulin folds of butyrophilin and the RING finger domain of ret finger protein. We recently reported several novel members of this family as well as previously undescribed protein families possessing the B30.2 domain. Many proteins have subsequently been found to possess this domain, including pyrin/marenostrin and the midline 1 (MID1) protein. Mutations in the B30.2 domain of pyrin/marenostrin are implicated in familial Mediterranean fever, and partial loss of the B30.2 domain of MID1 is responsible for Opitz G/BBB syndrome, characterized by developmental midline defects. In this study, we scrutinized the available sequence data bases for the identification of novel B30.2 domain proteins using highly sensitive database-searching tools. In addition, we discuss the chromosomal localization of genes in the B30.2 family, since the encoded proteins are likely to be involved in other forms of periodic fever, autoimmune, and genetic diseases.