Stabilization of a plasmid-encoded LacZ phenotype inBacillus subtilis

Recombinant plasmid pCED3 was structurally unstable inBacillus subtilis cultures grown in the presence of kanamycin to eliminate the effects of segregational instability. Analysis of 96 modified plasmids indicated that deletions in the plasmid occur at many different sites. The presence of plasmid pCED3 slowed the growth rate of theB. subtilis host. Cells that contained modified plasmids grew faster than the parental cells and took over the population. Two different methodologies were developed to reduce the cultural instability of the plasmid-directed LacZ⁺ phenotype. By growing the cells in a medium that supports a low growth rate, the growth rate ratio between modified and parental cells was reduced, resulting in a partial stabilization (40 generations) of the LacZ⁺ phenotype in the population [35]. Removal of a 4.77 kbEcoRI fragment (which consists primarily of the pBR322 replicon) from plasmid pCED3 produced a more stable plasmid derivative, designated pYS1. Cells harboring plasmid pYS1 grew faster than pCED3-bearing cells, although the level of activity of -galactosidase was similar in both strains. By combining the two approaches (i.e., growth of pYS1-bearing cells in a medium that supports low growth rate), the LacZ⁺ phenotype was stably maintained in the cell population for over 170 generations. Under these conditions, there was no detectable difference between the growth rates of cells bearing the pYS1 plasmid and further modified plasmids.     

Mating behavior of male deer ticksIxodes dammini (Acari: Ixodidae)

To analyze the sexual behavior of male black-legged deer ticks Ixodes dammini,we collected ticks infesting 202 white-tailed deer. On average, 17.7 males and 8.8 females infested each deer. Field-collected males copulated with a mean of 2.25 females, and virgin males mated with 2.4 females. On experimental hosts, males established sexual contact with feeding females and repelled other males, and about half remained paired after their mate detached. Engorged females continue to be receptive, and males mate more readily with them than with nonfed females. We conclude that male I. damminiare endowed with a repertoire of behaviors which favor an opportunistic mating before seeking a host and a preference for mating with feeding females on the host accompanied by tenacious mate guarding.     

Stable assembly of PsaE into cyanobacterial photosynthetic membranes is dependent on the presence of other accessory subunits of photosystem I

We studied assembly of the PsaE subunit of photosystem I into photosynthetic membranes of cyanobacterial mutant strains that lack specific photosystem I subunits. Radiolabeled PsaE was incubated with photosynthetic membranes, and their binding and assembly were assayed by resistance to removal by chaotropic agents and proteolytic digestion. PsaE incorporated into the wild-type membranes was resistant to these treatments. In the absence of PsaD, it was resistant to proteolytic digestion, but was removed by NaBr. When the membranes were isolated from a mutant strain in which the psaF and psaJ genes have been inactivated, PsaE assembled in vitro could not be removed. PsaE could associate with the membranes of the strain DF in which the psaD, psaJ and psaF genes have been mutated. However, the radiolabeled PsaE associated with these membranes was removed both by the proteolytic as well as by the chaotropic agents. Characterization of PsaE present in vivo revealed similar results. These observations suggest that PsaD and PsaF/J may interact with PsaE and stabilize it in the photosystem I complex.     

Molecular characterization of salt-stress-associated protein in citrus: protein and cDNA sequence homology to mammalian glutathione peroxidases

A gene encoding for a citrus salt-stress-associated protein (Cit-SAP) was cloned from Citrus sinensis salt-treated cell suspension. The gene, designated csa, was isolated from a cDNA expression library. The partial amino acid sequence of the protein, as well as that deduced from the nucleotide sequence of csa, revealed a considerable homology to mammalian glutathione peroxidase (GP), and to clone 6P229 from tobacco protoplasts. The increased expression of Cit-SAP in NaCl-treated cultured citrus cells and in citrus plants irrigated with saline water, and its similarity to GP, raise the possibility that one of the effects of salt stress in plants may be the increase of the level of free radicals.     

Drought,heat and salt stress induce the expression of a citrus homologue of an atypical late-embryogenesis Lea5 gene

In a search for genes that are induced in citrus cell suspension in response to salt stress, a cDNA clone with high homology to cotton Lea5 gene was isolated. Data base analysis of the protein deduced from the nucleotide sequence indicates that, like in cotton, the protein from citrus contains regions with significant hydropathic character. The gene, designated C-Lea5, is expressed in citrus leaves as well as cell suspension. The steady-state level of C-Lea5 is increased in cell suspension that is grown in the presence of 0.2 M NaCl. This phenomenon is also observed in leaves of citrus plants irrigated with NaCl and in citrus seedlings which are exposed to drought and heat stress. We suggest that the osmotic stress resulted from elevated level of salt is responsible for the increase in the level of C-Lea5.     

Phenology and polyploidy in annual Brachypodium species (Poaceae) along the aridity gradient in Israel

Local adaptation of plants along environmental gradients provides strong evidence for clinal evolution mediated by natural selection. Plants have developed diverse strategies to mitigate stress, for example, drought escape is a phenological strategy to avoid drought stress, while polyploidy was proposed as a genomic adaptation to stress. Polyploidy as an adaptation to aridity (an environmental parameter integrating temperature and precipitation) was previously documented in annual Brachypodium spp. (Poaceae) in the Western Mediterranean. Here, we examined whether polyploidy or phenology are associated with aridity in annual Brachypodium spp. along the aridity gradient in the Eastern Mediterranean. Using flow cytometry, we determined ploidy levels of plants from natural populations along the Israeli gradient, spanning ∼424 km from mesic Mediterranean to extreme desert climates. In a common garden we recorded time of seedling emergence, flowering and senescence. We tested whether the proportion of allotetraploids in the populations and phenological traits were associated with aridity. Contrary to a previous study in the Western Mediterranean, we found no effect of aridity on the proportion of allotetraploids and diploids within populations. Interestingly, phenology was associated with aridity: time of emergence was later, while flowering and senescence were earlier in desert plants. Our results indicate that in the Eastern Mediterranean, adaptation of Brachypodium to aridity is mediated mainly by phenology, rather than ploidy level. Therefore, we suggest that genome duplication is not the main driver of adaptation to environmental stress; rather, phenological change as a drought escape mechanism may be the major adaptation.     

Alignment free identification of clones in B cell receptor repertoires

Following antigenic challenge, activated B cells rapidly expand and undergo somatic hypermutation, yielding groups of clonally related B cells with diversified immunoglobulin receptors. Inference of clonal relationships based on the receptor sequence is an essential step in many adaptive immune receptor repertoire sequencing studies. These relationships are typically identified by a multi-step process that involves: (i) grouping sequences based on shared V and J gene assignments, and junction lengths and (ii) clustering these sequences using a junction-based distance. However, this approach is sensitive to the initial gene assignments, which are error-prone, and fails to identify clonal relatives whose junction length has changed through accumulation of indels. Through defining a translation-invariant feature space in which we cluster the sequences, we develop an alignment free clonal identification method that does not require gene assignments and is not restricted to a fixed junction length. This alignment free approach has higher sensitivity compared to a typical junction-based distance method without loss of specificity and PPV. While the alignment free procedure identifies clones that are broadly consistent with the junction-based distance method, it also identifies clones with characteristics (multiple V or J gene assignments or junction lengths) that are not detectable with the junction-based distance method.     

Ancestral haplotype reconstruction in endogamous populations using identity-by-descent

In this work we develop a novel algorithm for reconstructing the genomes of ancestral individuals, given genotype or sequence data from contemporary individuals and an extended pedigree of family relationships. A pedigree with complete genomes for every individual enables the study of allele frequency dynamics and haplotype diversity across generations, including deviations from neutrality such as transmission distortion. When studying heritable diseases, ancestral haplotypes can be used to augment genome-wide association studies and track disease inheritance patterns. The building blocks of our reconstruction algorithm are segments of Identity-By-Descent (IBD) shared between two or more genotyped individuals. The method alternates between identifying a source for each IBD segment and assembling IBD segments placed within each ancestral individual. Unlike previous approaches, our method is able to accommodate complex pedigree structures with hundreds of individuals genotyped at millions of SNPs.We apply our method to an Old Order Amish pedigree from Lancaster, Pennsylvania, whose founders came to North America from Europe during the early 18th century. The pedigree includes 1338 individuals from the past 12 generations, 394 with genotype data. The motivation for reconstruction is to understand the genetic basis of diseases segregating in the family through tracking haplotype transmission over time. Using our algorithm thread, we are able to reconstruct an average of 224 ancestral individuals per chromosome. For these ancestral individuals, on average we reconstruct 79% of their haplotypes. We also identify a region on chromosome 16 that is difficult to reconstruct—we find that this region harbors a short Amish-specific copy number variation and the gene HYDIN. thread was developed for endogamous populations, but can be applied to any extensive pedigree with the recent generations genotyped. We anticipate that this type of practical ancestral reconstruction will become more common and necessary to understand rare and complex heritable diseases in extended families.     

Comprehensive Mapping of Regional Expression of the Clock Protein PERIOD2 in Rat Forebrain across the 24-h Day

In mammals, a light-entrainable clock located in the suprachiasmatic nucleus (SCN) regulates circadian rhythms by synchronizing oscillators throughout the brain and body. Notably, the nature of the relation between the SCN clock and subordinate oscillators in the rest of the brain is not well defined. We performed a high temporal resolution analysis of the expression of the circadian clock protein PERIOD2 (PER2) in the rat forebrain to characterize the distribution, amplitude and phase of PER2 rhythms across different regions. Eighty-four LEW/Crl male rats were entrained to a 12-h: 12-h light/dark cycle, and subsequently perfused every 30 min across the 24-h day for a total of 48 time-points. PER2 expression was assessed with immunohistochemistry and analyzed using automated cell counts. We report the presence of PER2 expression in 20 forebrain areas important for a wide range of motivated and appetitive behaviors including the SCN, bed nucleus, and several regions of the amygdala, hippocampus, striatum, and cortex. Eighteen areas displayed significant PER2 rhythms, which peaked at different times of day. Our data demonstrate a previously uncharacterized regional distribution of rhythms of a clock protein expression in the brain that provides a sound basis for future studies of circadian clock function in animal models of disease.