Studies of ribosomal proteins of yeast species and their hybrids gel electrophoresis and immunochemical cross-reactions

Summary The cytoplasmic ribosomal proteins (r-proteins) of seventeen yeast species of the genera Saccharomyces and Kluyveromyces were analyzed by one-dimensional gradient polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The electrophoretic patterns of cytoplasmic r-proteins from different species display extensive differences in both the 40S and the 60S subunit. Relatedness of species suggested by r-protein patterns correlates well with that based on DNA/DNA homology (Bicknell and Douglas 1970). Immunochemical cross-reactions and antibiotic susceptibility tests were also used to compare different species.Analyses of r-proteins from two different interspecific hybrids showed that their ribosomes were hybrid, containing r-proteins from both parents. These findings are discussed in relation to the evolution of yeast species and the regulation of expression of r-proteins in cucaryotes.     

Development and assessment of a landscape-scale ecological threat index for the Lower Colorado River Basin

Anthropogenic disturbances impact freshwater biota but are rarely incorporated into conservation planning due to the difficulties in quantifying threats. There is currently no widely accepted method to quantify disturbances, and determining how to measure threats to upstream catchments using disturbance metrics can be time consuming and subjective. We compared four watershed-scale ecological threat indices for the Lower Colorado River Basin (LCRB) using landscape-level threats of land use (e.g., agricultural and urban lands), waterway development and diversions (e.g., number of canals, dams), and human development (e.g., road and railroads density, pollution sites). The LCRB is an ideal region to assess ecological threat indices because of the increasing need for conservation to ensure the persistence of native fishes in highly altered habitat. Each threat was measured for severity (i.e., level of influence on the upstream watershed) and frequency throughout each watershed; both severity and frequency were measured using two different methods. Severity values were based either on peer-reviewed literature and weighted in accordance to their published ecological impact, or assumed equal severity across stressors. Threat frequency was calculated according to either the presence/absence of each stressor, or on the relative density of each stressor in the watershed. Each measure of severity was combined with a measure of frequency, creating four ecological threat indices, and transformed to a 0–100 scale. Threat indices were highly correlated (slopes of 0.94–1.63; R² of 0.82–0.98), and were highest for watersheds close to urban centers, including Phoenix, Tucson, and Flagstaff, Arizona, and Las Vegas, Nevada. Road crossings and density appeared to be the most influential stressors in the index, but the removal of any individual stressor only changed the index by <5.1 units. Our results indicate that a simpler index with less subjectivity (i.e., presence/absence of a stressor in a watershed) provides similar results to the more subjective measure of threats (i.e., peer-reviewed threat severity). Because these threats have been linked to ecological health, the development of the index should be a useful tool to identify regions of greatest potential threat to aquatic biota and can aid in conservation planning for the Lower Colorado River Basin.     

Temperature dependence of collagen fluorescence.

Dermal collagens have several fluorescent moieties in the UV and visible spectral regions that may serve as molecular probes of collagen. We studied the temperature-dependence of a commercial calf skin collagen and acid-extracted Skh-1 hairless mouse collagen at temperatures from 9 degrees C to 60 degrees C for excitation/emission wavelengths 270/305 nm (tyrosine), 270/360 nm (excimer-like aggregated species), 325/400 nm (dityrosine) and 370/450 nm (glycation adduct). L-tyrosine (1 x 10(-5) M in 0.5 M HOAc) acted as a "reference compound" devoid of any collagen structural effects. In general, the fluorescence efficiency of these fluorophores decreases with increasing temperature. Assuming that rate constant for fluorescence deactivation has the form k(d)(T) = k(d) degrees exp (-DeltaE/RT), an Arrhenius plot of log[(1/Phi) - 1] vs. 1/T affords a straight line whose (negative) slope is proportional to the activation energy, DeltaE, of the radiationless process(es) that compete with fluorescence. Because it is difficult to accurately measure Phi(f) for collagen-bound fluorophores, we derived an approximate formula for an activation parameter, DeltaE*, evaluated from an Arrhenius-like plot of log 1/I(N)vs. 1/T, (1/I(N)vs. is the reciprocal normalized fluorescence intensity). Tyrosine in dilute solution affords a linear Arrhenius plot in both of the above cases. Using the known value of Phi(f) = 0.21 for free tyrosine at room temperature, we determined that DeltaE* is accurate to approximately 25% in the present instance. Collagen curves are non-linear, but they are quasi-linear below approximately 20 degrees C, where the helical form predominates. Values of DeltaE* determined from the data at T < 20 degrees C ranged from 6.2-8.4 kJ mol(-1) (1.5-2.0 kcal mol(-1)) for mouse collagen and 10.3-11.4 kJ mol(-1) (2.5-2.7 kcal mol(-1)) for calf skin collagen, consistent with collisional deactivation of the fluorescent state via thermally enhanced molecular vibrations and rotations. Above 20 degrees C, log 1/I(N)vs. 1/T plots from Skh-1 hairless mouse collagen are concave-downward, suggesting that fluorescence deactivation from the denatured coil has a significant temperature-independent component. For calf skin collagen, these plots are concave-upward, suggesting an increase in activation energy above Tm. These results suggest that collagen backbone and supramolecular structure can influence the temperature dependence of the bound fluorophores, indicating the future possibility of using activation data as a probe of supramolecular structure and conformation.     

Effects of DNase production, plasmid size, and restriction barriers on transformation of Vibrio cholerae by electroporation and osmotic shock

Attempts to transform wild type strains of V. cholerae with plasmid DNA by traditional osmotic shock methods were not successful. A mutant of V. cholerae that was deficient in extracellular DNase was transformed with plasmid DNA by osmotic shock, demonstrating directly that extracellular DNase is a major barrier to transformation of V. cholerae. Transformation of wild type and DNase-negative strains of V. cholerae was accomplished by electroporation. Efficiency of transformation by electroporation increased with field strength, decreased with plasmid size, and was relatively insensitive to changes in the electrolyte composition of the buffer as long as isotonic sucrose was present. Host-controlled modification/restriction systems also affected transformation efficiency in V. cholerae.     

Altered differentiation and clustering of Sertoli cells in transgenic mice showing a Sertoli cell specific knockout of the connexin 43 gene

Histological analysis revealed that Sertoli cell specific knockout of the predominant testicular gap junction protein connexin 43 results in a spermatogenic arrest at the level of spermatogonia or Sertoli cell-only syndrome, intratubular cell clusters and still proliferating adult Sertoli cells, implying an important role for connexin 43 in the Sertoli and germ cell development. This study aimed to determine the (1) Sertoli cell maturation state, (2) time of occurrence and (3) composition, differentiation and fate of clustered cells in knockout mice. Using immunohistochemistry connexin 43 deficient Sertoli cells showed an accurate start of the mature markers androgen receptor and GATA-1 during puberty and a vimentin expression from neonatal to adult. Expression of anti-Muellerian hormone, as a marker of Sertoli cell immaturity, was finally down-regulated during puberty, but its disappearance was delayed. This observed extended anti-Müllerian hormone synthesis during puberty was confirmed by western blot and Real-Time PCR and suggests a partial alteration in the Sertoli cell differentiation program. Additionally, Sertoli cells of adult knockouts showed a permanent and uniform expression of GATA-1 at protein and mRNA level, maybe caused by the lack of maturing germ cells and missing negative feedback signals. At ultrastructural level, basally located adult Sertoli cells obtained their mature appearance, demonstrated by the tripartite nucleolus as a typical feature of differentiated Sertoli cells. Intratubular clustered cells were mainly formed by abnormal Sertoli cells and single attached apoptotic germ cells, verified by immunohistochemistry, TUNEL staining and transmission electron microscopy. Clusters first appeared during puberty and became more numerous in adulthood with increasing cell numbers per cluster suggesting an age-related process. In conclusion, adult connexin 43 deficient Sertoli cells seem to proliferate while maintaining expression of mature markers and their adult morphology, indicating a unique and abnormal intermediate phenotype with characteristics common to both undifferentiated and differentiated Sertoli cells.     

Three new species of Hagnagora Druce, 1885 (Lepidoptera,Geometridae, Larentiinae) from Ecuador and Costa Rica and a concise revision of the genus

Three new Hagnagora Druce species (Geometridae, Larentiinae) are described: Hagnagora richardi Brehm, sp. n. from Ecuador, Hagnagora hedwigae Brehm, sp. n. from Ecuador, and Hagnagora mirandahenrichae Brehm, sp. n. from Costa Rica. A checklist of taxa assigned to Hagnagora is provided. Hagnagora is provisionally divided into six clades: the anicata clade (6 species), the buckleyi clade (3 species), the croceitincta clade (3 species), the ephestris clade (3 species), the mortipax clade (4 species) and Hagnagora subrosea (1 species). Two taxa are revived from synonymy: Hagnagora catagrammina Druce, stat. rev. and Hagnagora luteoradiata Thierry-Mieg, stat. rev. Two taxa are reinstated from subspecies to species level: Hagnagora acothysta Schaus, stat. rev. and Hagnagora jamaicensis Schaus, stat. rev. Four taxa are provisionally removed from Hagnagora: “Hagnagora” ignipennis, “Hagnagora” mesenata, “Hagnagora” vittata, and “Hagnagora” ceraria. After these changes, the genus Hagnagora now comprises 20 valid species.     

Efficient and error-free fluorescent gene tagging in human organoids without double-strand DNA cleavage

CRISPR-associated nucleases are powerful tools for precise genome editing of model systems, including human organoids. Current methods describing fluorescent gene tagging in organoids rely on the generation of DNA double-strand breaks (DSBs) to stimulate homology-directed repair (HDR) or non-homologous end joining (NHEJ)-mediated integration of the desired knock-in. A major downside associated with DSB-mediated genome editing is the required clonal selection and expansion of candidate organoids to verify the genomic integrity of the targeted locus and to confirm the absence of off-target indels. By contrast, concurrent nicking of the genomic locus and targeting vector, known as in-trans paired nicking (ITPN), stimulates efficient HDR-mediated genome editing to generate large knock-ins without introducing DSBs. Here, we show that ITPN allows for fast, highly efficient, and indel-free fluorescent gene tagging in human normal and cancer organoids. Highlighting the ease and efficiency of ITPN, we generate triple fluorescent knock-in organoids where 3 genomic loci were simultaneously modified in a single round of targeting. In addition, we generated model systems with allele-specific readouts by differentially modifying maternal and paternal alleles in one step. ITPN using our palette of targeting vectors, publicly available from Addgene, is ideally suited for generating error-free heterozygous knock-ins in human organoids.

A major downside of double-strand break-mediated genome editing is the need to verify the genomic integrity of the targeted locus and confirm the absence of off-target indels. This study shows that in-trans paired nicking is a mutation-free CRISPR strategy to introduce precise knock-ins into human organoids; its genomic fidelity allows all knock-in cells to be pooled, accelerating the establishment of new organoid models.     

Taxonomic and functional homogenization of an endemic desert fish fauna

Aim The highly endemic fishes of the arid Southwest USA have been heavily impacted by human activities resulting in one of the most threatened fish faunas in the world. The aim of this study was to examine the patterns and drivers of taxonomic and functional beta diversity of freshwater fish in the Lower Colorado River Basin across the 20th century. Location Lower Colorado River Basin (LCRB). Methods The taxonomic and functional similarities of watersheds were quantified to identify patterns of biotic homogenization or differentiation over the period 1900–1999. Path analysis was used to identify the relative influence of dam density, urban land use, precipitation regimes and non‐native species richness on observed changes in fish faunal composition. Results The fish fauna of the LCRB has become increasingly homogenized, both taxonomically (1.1% based on β_sim index) and functionally (6.2% based on Bray–Curtis index), over the 20th century. The rate of homogenization varied substantially; range declines of native species initially caused taxonomic differentiation (?7.9% in the 1960s), followed by marginal homogenization (observed in the 1990s) in response to an influx of non‐native species introductions. By contrast, functional homogenization of the basin was evident considerably earlier (in the 1950s) because of the widespread introduction of non‐native species sharing similar suites of biological traits. Path analysis revealed that both taxonomic and functional homogenization were positively related to the direct and indirect (facilitation by dams and urbanization) effects of non‐native species richness. Main conclusions Our study simultaneously examines rates of change in multiple dimensions of the homogenization process. For the endemic fish fauna of the LCRB, we found that the processes of taxonomic and functional homogenization are highly dynamic over time, varying both in terms of the magnitude and rate of change over the 20th century.     

Identification and characterization of the aspartate chemosensory receptor of Campylobacter jejuni

Campylobacter jejuni is a highly motile bacterium that responds via chemotaxis to environmental stimuli to migrate towards favourable conditions. Previous in silico analysis of the C. jejuni strain NCTC11168 genome sequence identified 10 open reading frames, tlp1‐10, that encode putative chemosensory receptors. We describe the characterization of the role and specificity of the Tlp1 chemoreceptor (Cj1506c). In vitro and in vivo models were used to determine if Tlp1 had a role in host colonization. The tlp1^‐ isogenic mutant was more adherent in cell culture, however, showed reduced colonization ability in chickens. Specific interactions between the purified sensory domain of Tlp1 and l ‐aspartate were identified using an amino acid array and saturation transfer difference nuclear magnetic resonance spectroscopy. Chemotaxis assays showed differences between migration of wild‐type C. jejuni cells and that of a tlp1^‐ isogenic mutant, specifically towards aspartate. Furthermore, using yeast two‐hybrid and three‐hybrid systems for analysis of protein–protein interactions, the cytoplasmic signalling domain of Tlp1 was found to preferentially interact with CheV, rather than the CheW homologue of the chemotaxis signalling pathway; this interaction was confirmed using immune precipitation assays. This is the first identification of an aspartate receptor in bacteria other than Escherichia coli and Salmonella enterica serovar Typhimurium.