Mitochondrial DNA Sequence Divergence among Meloidogyne incognita,Romanomermis culicivorax,Ascaris suum,and Caenorhabditis elegans

Mitochondrial DNA sequences were obtained from the NADH dehydrogenase subunit 3 (ND3), large rRNA, and cytochrome b genes from Meloidogyne incognita and Romanomermis culicivorax. Both species show considerable genetic distance within these same genes when compared with Caenorhabditis elegans or Ascaris suum, two species previously analyzed. Caenorhabditis, Ascaris, and Meloidogyne were selected as representatives of three subclasses in the nematode class Secernentea: Rhabditia, Spiruria, and Diplogasteria, respectively. Romanomermis served as a representative out-group of the class Adenophorea. The divergence between the phytoparasitic lineage (represented by Meloidogyne) and the three other species is so great that virtually every variable position in these genes appears to have accumulated multiple mutations, obscuring the phylogenetic information obtainable from these comparisons. The 39 and 42% amino acid similarity between the M. incognita and C. elegans ND3 and cytochrome b coding sequences, respectively, are approximately the same as those of C. elegans-mouse comparisons for the same genes (26 and 44%). This discovery calls into question the feasibility of employing cloned C. elegans probes as reagents to isolate phytoparasitic nematode genes. The genetic distance between the phytoparasitic nematode lineage and C. elegans markedly contrasts with the 79% amino acid similarity between C. elegans and A. suum for the same sequences. The molecular data suggest that Caenorhabditis and Ascaris belong to the same subclass.     

Fungal catabolic gene regulation: Molecular genetic analysis of theamdS gene ofAspergillus nidulans

Aspergillus nidulans is an excellent experimental organism for the study of gene regulation. Genetic and molecular analyses oftrans-acting andcis-acting mutations have revealed a complex pattern of regulation involving multiple independent controls. Expression of theamdS gene is regulated by thefacB andamdA genes which encode positively acting regulatory proteins mediating a major and a minor form of acetate induction respectively. The product of theamdR gene mediates omega amino acid induction ofamdS. The binding sites for each of these proteins have been localised throughamdS cis-acting mutations which specifically affect the interaction with the regulatory protein. The global controls of nitrogen metabolite repression and carbon catabolite repression regulate the expression of many catabolic genes, includingamdS. Nitrogen control is exerted through the positively actingareA gene product and carbon control is dependent on thecreA gene product. Each of the characterized regulatory genes encodes a DNA-binding protein which recognises particular sequences in theamdS promoter to activate or repress gene expression. In addition, there is evidence for other genetically uncharacterised proteins, including a CCAAT-binding complex, which interact with the 5 region of theamdS gene.     

A novel molecular fingerprint probe based on the endogenous avian retroviral element (EAV) of chickens

We have developed a novel molecular probe that is useful for DNA fingerprint analysis in chickens. The probe is based on the middle-repetitive, chicken endogenous retroviral (EAV) element. It consists of 1503 bp of the 3′ portion of the EAV element, extending from the downstream end of the envelope gene to the beginning of the downstream long terminal repeat (LTR). Unlike other probes that are currently in use for fingerprint analysis with chicken DNA, the EAV-based probe works well at normal levels of stringency, and with standard hybridization buffers. Digestion of chicken genomic DNA with a variety of restriction enzymes routinely yields up to 30 resolvable bands per bird in the 500 bp to 20kbp range. In order to test the efficacy of the EAV-based fingerprint probe, we have used it to estimate the degree of inbreeding in the inbred WG strain of White Leghorns. We find that the estimates derived with the EAV probe are very similar to those reported previously for the WG strain. These results suggest that molecular probes based on endogenous retroviruses and other middle-repetitive DNA elements should be useful for fingerprint analysis in chickens, and in vertebrates in general.     

DNA/DNA hybridization studies of carnivorous marsupials. III. Relationships among species ofDidelphis (Didelphidae)

We report three sets of DNA hybridization experiments conducted to determine relationships among species ofDidelphis (D. albiventris, D. marsupialis, D. virginiana). The 1989 and 1991 sets had fewer replicates per cell than the 1990 series (3.4 and 5.4 vs 9), but in 1991 we distinguished two populations ofD. marsupialis and utilized several individuals for each heterologous comparison. BothPhilander opossum andLutreolina crassicaudata were used as outgroups in 1989, but onlyLutreolina was included in subsequent sets. For each set, we calculated all four standard indices of thermal stability (T _mode,T _m,T ₅₀ H, and NPH) and constructed trees by least-squares (FITCH) and neighbor-joining methods, both before and after correction for asymmetric reciprocal cell values. Subsets of the 1989 data lacking eitherPhilander orLutreolina were analyzed similarly. To explore measurement imprecision, the corrected and uncorrected matrices for each of the four indices were bootstrapped 100 times for the 1989 set and subsets and 1000 times for the 1990 and 1991 sets. Again, for the 1990 and 1991 data, an additional 100 bootstrapped distances were fitted to user trees representing the three possible pairings ofDidelphis spp. to determine the significance of the FITCH branch lengths. The successive experimental sets generated increasingly consistent evidence for pairingD. marsupialis withD. albiventris. The 1989 experiments involved just 85 comparisons, and only T _mode's pairedD. marsupialis withD. albiventris (at bootstrap percentages of 70% or above), but did so whetherPhilander orLutreolina or both were included as the outgroup(s). FITCH and neighbor-joining trees had identical topologies for T _mode's but sometimes differed for the other measures. In contrast, all but one of the corresponding FITCH and neighborjoining trees matched for the 1990 and 1991 data, and three of the four distance measures (T _mode, T _m, and T ₅₀ H) unitedD. marsupialis withD. albiventris at bootstrap percentages averaging 81%; NPH gave a different result for 1990, associatingD. marsupialis withD. virginiana. Further, all but 2 of the 16 matrices for 1990 and 1991 gave mean bootstrapped branch lengths for a consensus pairing that were positive and at least one standard deviation from zero, despite the very short internodes recovered. These results illustrate that the potential of DNA hybridization for resolving very close relationships depends on both the index and the experimental design employed. We conclude that of the three species,D. albiventris andD. marsupialis shared a more recent common ancestor and estimate thatDidelphis spp. have diverged at about 0.39% in nucleotide sequence per myr.Deceased.     

A molecular perspective on the phylogeny of placental mammals based on mitochondrial 12S rDNA sequences,with special reference to the problem of the Paenungulata

Part of the 12S rDNA gene was amplified and sequenced for 11 placental mammals, 3 marsupials, and 2 monotremes. Multiple alignments for these sequences and nine additional placental sequences taken from GenBank were obtained using CLUSTAL. Phylogenetic analyses were performed using standard parsimony, transversion parsimony, and Lake's method of invariants. All of our analyses uniteLoxodontia withDugong. Procavia, in turn, is a sister group to these taxa, thus supporting the monophyly of the Paenungulata. Perissodactyls are a sister group to paenungulates when transitions and transversions are both included but not when transitions are omitted. Likewise, cetaceans are a sister group to artiodactyls on minimum length trees under standard parsimony but not under transversion parsimony. Rodent monophyly and bat monophyly also receive mixed support, as does a putative alliance between primates and lagomorphs. Interestingly, the percentage divergence between the echidna and the platypus is less than for the rat and mouse.     

绿色木霉组蛋白去乙酰化酶编码基因TvRpd3在木霉生物防治作用中的功能分析

【目的】本文借助基因编辑技术在具有生物防治潜力的绿色木霉(Trichoderma viride)中敲除组蛋白去乙酰化酶编码基因TvRpd3,来研究TvRpd3基因及其编码蛋白在提高木霉病原菌拮抗能力中的作用。【方法】利用融合PCR和同源重组策略构建了TvRpd3基因缺失的突变菌株,通过对峙培养、表型观察、免疫组化检测、代谢组学分析等系统比较TvRpd3基因敲除前后菌株的组蛋白乙酰化修饰水平、次级代谢产物合成、病原菌拮抗能力以及田间防治效果等。【结果】与野生型菌株相比,缺失TvRpd3基因的木霉工程菌(?TvRpd3)对多种病原菌表现出了更强的对峙抑制效果,其所产的发酵液对小麦白粉病、烟草黑胫病和番茄枯萎病的防治效果分别提高了62.27%、57.45%和70.71%。同时,敲除TvRpd3基因也显著改变了木霉工程菌所产次级代谢产物的种类和产量,抗生性物质的产量大幅提高。【结论】绿色木霉TvRpd3基因及其介导的组蛋白乙酰化修饰在提高绿色木霉生物防治中起着重要作用。     

N-glycosylation induced changes in tau protein dynamics reveal its role in tau misfolding and aggregation: A microsecond long molecular dynamics study

Various posttranslational modifications like hyperphosphorylation, O-GlcNAcylation, and acetylation have been attributed to induce the abnormal folding in tau protein. Recent in vitro studies revealed the possible involvement of N-glycosylation of tau protein in the abnormal folding and tau aggregation. Hence, in this study, we performed a microsecond long all atom molecular dynamics simulation to gain insights into the effects of N-glycosylation on Asn-359 residue which forms part of the microtubule binding region. Trajectory analysis of the stimulations coupled with essential dynamics and free energy landscape analysis suggested that tau, in its N-glycosylated form tends to exist in a largely folded conformation having high beta sheet propensity as compared to unmodified tau which exists in a large extended form with very less beta sheet propensity. Residue interaction network analysis of the lowest energy conformations further revealed that Phe378 and Lys353 are the functionally important residues in the peptide which helped in initiating the folding process and Phe378, Lys347, and Lys370 helped to maintain the stability of the protein in the folded state.     

A new approach for extracting information from protein dynamics

Increased ability to predict protein structures is moving research focus towards understanding protein dynamics. A promising approach is to represent protein dynamics through networks and take advantage of well-developed methods from network science. Most studies build protein dynamics networks from correlation measures, an approach that only works under very specific conditions, instead of the more robust inverse approach. Thus, we apply the inverse approach to the dynamics of protein dihedral angles, a system of internal coordinates, to avoid structural alignment. Using the well-characterized adhesion protein, FimH, we show that our method identifies networks that are physically interpretable, robust, and relevant to the allosteric pathway sites. We further use our approach to detect dynamical differences, despite structural similarity, for Siglec-8 in the immune system, and the SARS-CoV-2 spike protein. Our study demonstrates that using the inverse approach to extract a network from protein dynamics yields important biophysical insights.     

Higher-order interactions of Bcr-Abl can broaden chronic myeloid leukemia (CML) drug repertoire

Bcr-Abl, a nonreceptor tyrosine kinase, is associated with leukemias, especially chronic myeloid leukemia (CML). Deletion of Abl's N-terminal region, to which myristoyl is linked, renders the Bcr-Abl fusion oncoprotein constitutively active. The substitution of Abl's N-terminal region by Bcr enables Bcr-Abl oligomerization. Oligomerization is critical: it promotes clustering on the membrane, which is essential for potent MAPK signaling and cell proliferation. Here we decipher the Bcr-Abl specific, step-by-step oligomerization process, identify a specific packing surface, determine exactly how the process is structured and identify its key elements. Bcr's coiled coil (CC) domain at the N-terminal controls Bcr-Abl oligomerization. Crystallography validated oligomerization via Bcr-Abl dimerization between two Bcr CC domains, with tetramerization via tight packing between two binary assemblies. However, the structural principles guiding Bcr CC domain oligomerization are unknown, hindering mechanistic understanding and drugs exploiting it. Using molecular dynamics (MD) simulations, we determine that the binary complex of the Bcr CC domain serves as a basic unit in the quaternary complex providing a specific surface for dimer–dimer packing and higher-order oligomerization. We discover that the small α1-helix is the key. In the binary assembly, the helix forms interchain aromatic dimeric packing, and in the quaternary assembly, it contributes to the specific dimer–dimer packing. Our mechanism is supported by the experimental literature. It offers the key elements controlling this process which can expand the drug discovery strategy, including by Bcr CC-derived peptides, and candidate residues for small covalent drugs, toward quenching oligomerization, supplementing competitive and allosteric tyrosine kinase inhibitors.     

Drosophila acidic ribosomal protein rpA2: Sequence and characterization

A cDNA encoding the Drosophila melanogaster acidic ribosomal protein rpA2 was cloned and sequenced. rpA2 is homologous to the Artemia salina acidic ribosomal protein eL12′. In situ hybridization to salivary gland polytene chromosomes localizes the rpA2 gene to band 21C. It is a single copy gene, with an mRNA of 0.8 kb. Two-dimensional gel electrophoresis of Drosophila ribosomal proteins followed by immuno-blotting showed that the rpA2 protein has an apparent relative mobility in SDS of 17 kD and an isoelectric point less than pH 5.0. Although the Drosophila gene rp21C may be the same as rpA2, the reported sequences differ. Comparisons of the aligned nucleotide sequences coding for the acidic ribosomal proteins rpA1 and rpA2 of Drosophila with those of other eukaryotes support the view of two separate, though closely related, groups of acidic proteins. Comparison with the Artemia homologues suggests that nucleotide identity may have been conserved by some constraint that acts in addition to the requirement for substantial similarity of amino acid sequences. © 1993 Wiley-Liss, Inc.