Bypass of DNA lesions generated during anticancer treatment with cisplatin by DNA polymerase eta

DNA polymerase eta (Pol eta) is a eukaryotic lesion bypass polymerase that helps organisms to survive exposure to ultraviolet (UV) radiation, and tumor cells to gain resistance against cisplatin-based chemotherapy. It allows cells to replicate across cross-link lesions such as 1,2-d(GpG) cisplatin adducts (Pt-GG) and UV-induced cis-syn thymine dimers. We present structural and biochemical analysis of how Pol eta copies Pt-GG-containing DNA. The damaged DNA is bound in an open DNA binding rim. Nucleotidyl transfer requires the DNA to rotate into an active conformation, driven by hydrogen bonding of the templating base to the dNTP. For the 3'dG of the Pt-GG, this step is accomplished by a Watson-Crick base pair to dCTP and is biochemically efficient and accurate. In contrast, bypass of the 5'dG of the Pt-GG is less efficient and promiscuous for dCTP and dATP as a result of the presence of the rigid Pt cross-link. Our analysis reveals the set of structural features that enable Pol eta to replicate across strongly distorting DNA lesions.     

Comparative genome and proteome analysis of Anopheles gambiae and Drosophila melanogaster

Comparison of the genomes and proteomes of the two diptera Anopheles gambiae and Drosophila melanogaster, which diverged about 250 million years ago, reveals considerable similarities. However, numerous differences are also observed; some of these must reflect the selection and subsequent adaptation associated with different ecologies and life strategies. Almost half of the genes in both genomes are interpreted as orthologs and show an average sequence identity of about 56%, which is slightly lower than that observed between the orthologs of the pufferfish and human (diverged about 450 million years ago). This indicates that these two insects diverged considerably faster than vertebrates. Aligned sequences reveal that orthologous genes have retained only half of their intron/exon structure, indicating that intron gains or losses have occurred at a rate of about one per gene per 125 million years. Chromosomal arms exhibit significant remnants of homology between the two species, although only 34% of the genes colocalize in small "microsyntenic" clusters, and major interarm transfers as well as intra-arm shuffling of gene order are detected.     

Ordered and dynamic assembly of single spliceosomes

The spliceosome is the complex macromolecular machine responsible for removing introns from precursors to messenger RNAs (pre-mRNAs). We combined yeast genetic engineering, chemical biology, and multiwavelength fluorescence microscopy to follow assembly of single spliceosomes in real time in whole-cell extracts. We find that individual spliceosomal subcomplexes associate with pre-mRNA sequentially via an ordered pathway to yield functional spliceosomes and that association of every subcomplex is reversible. Further, early subcomplex binding events do not fully commit a pre-mRNA to splicing; rather, commitment increases as assembly proceeds. These findings have important implications for the regulation of alternative splicing. This experimental strategy should prove widely useful for mechanistic analysis of other macromolecular machines in environments approaching the complexity of living cells.     

Evaluation of First-Feeding Regimens for Larval Nassau Grouper Epinephelus straitus and Preliminary, Pilot-Scale Culture through Metamorphosis

Two 10-day hatchery experiments were conducted to evaluate s-type (Hawaiian strain) and ss-type (Thailand strain) rotifers Brachionus plicatilis and cryogenically preserved oyster Crassostrea gigas trochophores as first feeds for larval Nassau grouper Epinephelus striatus. Newly hatched grouper larvae were reared at densities of 11.2–20.8/L in 500-L tanks at 36–38 ppt salinity, 25–26 C, and under a 11-h light: 13-h dark photoperiod. Beginning on day 2 posthatching (d2ph), prey were maintained at a density of 20 individuals/mL, while phytoplankton (Nanochloropsis oculata) was maintained at 500 × 10³ cells/mL. In experiment 1, survival and growth were higher (P < 0.05) for fish fed small s-type rotifers (mean lorica length = 117 μm; fish survival = 7.96%) selected by sieving than for fish fed non-selected rotifers (mean lorica length = 161 μm; fish survival = 2.13%). These results demonstrated the advantage of small prey size and suggested that super-small (ss-type) rotifer strains would be beneficial. In experiment 2, three feeding regimens were compared: 1) ss-type rotifers (mean lorica length = 147 μm); 2) oyster trochophores (mean diameter = 50 μm) gradually replaced by ss-type rotifers from d5ph; and 3) a mixed-prey teatment of 50% oyster trochophores and 50% ss-type rotifers. Survival was higher (P < 0.05) for larvae fed mixed prey (15.6%) than for those fed rotifers (9.73%) or trochophores and rotifers in sequence (2.55%), which also showed the slowest growth. Oyster trochophores, although inadequate when used exclusively, enhanced survival when used in combination with rotifers, possibly by improving size selectivity and dietary quality. In a pilot-scale trial, larvae were cultured through metamorphosis in two 33.8-m³ outdoor tanks. Fertilized eggs were stocked at a density of 10 eggs/L and larvae were fed ss-type rotifers from d2ph-d20ph, newly hatched Artemia from d15ph-d18ph, 1-d-old Artemia nauplii from d18ph-d62ph. Survival on d62ph was 1.17%, with a total of 5,651 post-metamorphic juveniles produced.     

The influence of scale and the spatial characteristics of landscapes on land-cover mapping using remote sensing

Statistical analyses provide a means for assessing relationships between landscape spatial pattern and errors in the estimates of cover-type proportions as land-cover data are aggregated to coarser scales. Results from a multiple-linear regression model suggest that as patch sizes, variance/mean ratio, and initial proportions of cover types increase, the proportion error moves in a positive direction and is governed by the interaction of the spatial characteristics and the scale of aggregation. However, the standard linear model does not account for the different directions of scale-dependent proportion error since some classes become larger and others become smaller as the scene is aggregated. Addition of indicator variables representing class-type significantly improves the performance by allowing the model to respond differently to different classes. A regression tree model provides a much simpler fit to the complex scaling behavior through an interaction between patch size and aggregation scale. An understanding of the relationships between landscape pattern, scale, and proportion error may advance methods for correcting land-cover area estimates. Such methods could also facilitate high-resolution calibration and validation of coarse-scale remote-sensing-based land-cover mapping algorithms. Ongoing initiatives to produce global land-cover datasets from remote sensing, such as efforts within the IGBP and the EOS MODIS Land-Team, include significant emphasis on high level calibration and validation activities of this nature.     

A comparison of growth rates of normal thoroughbred foals and foals diagnosed with cervical vertebral malformation

The objectives of this study were to compare the growth rates of normal thoroughbred foals to previously reported growth rates¹ and to compare growth rates of normal foals with foals diagnosed with cervical vertebral malformation (CVM). Fifty-six foals from two foals crops were used, and eight of these foals were diagnosed with CVM. Growth rate analysis of the CVM foals had to be restricted to the period prior to diagnosis because once these foals were diagnosed, they were treated differently from the normal foals. Body measurements taken at 7 day intervals were body weight, wither height, hip height, and heart girth. For statistical analysis, data were broken down into 30 day intervals. Results showed that the growth rates for normal thoroughbred foals have not changed in the past fifteen years. The CVM foals tended to be heavier and taller during some time intervals than the normal foals, but there were no significant differences between the two groups in any of the skeletal growth measurements. Body weight gain was faster in CVM foals from 31–60 d (p<.01), 121–150 d (p<.01), and 211–240 d (P –.05).     

Immune response to recombinant Escherichia coli Iss protein in poultry

Colibacillosis accounts for significant losses to the poultry industry, and control efforts are hampered by limited understanding of the mechanisms used by avian pathogenic Escherichia coli (APEC) to cause disease. We have found that the presence of the increased serum survival gene (iss) is strongly associated with APEC but not with commensal E. coli, making iss, and the protein it encodes (Iss), candidate targets of colibacillosis control procedures. To assess the potential of Iss to elicit a protective response in chickens against APEC challenge, Iss fusion proteins were produced and administered subcutaneously to four groups of 2-wk-old specific-pathogen-free leghorn chickens. At 4 wk postimmunization, birds were challenged with APEC from serogroups 02 and 078 via intramuscular injection. At 2 wk postchallenge, birds were necropsied, and lesions consistent with colibacillosis were scored. Also, sera were collected from the birds pre- and postimmunization, and antibody titers to Iss were determined. Immunized birds produced a humoral response to Iss, and they had significantly lower lesion scores than the unimmunized control birds following challenge with both APEC strains. Birds that received the smallest amount of immunogen had the lowest lesion scores. Although further study will be needed to confirm the value of Iss as an immunoprotective antigen, these preliminary data suggest that Iss may have the potential to elicit significant protection in birds against heterologous E. coli challenge.