scholarly journals The effects of truncations of the small subunit on m-calpain activity and heterodimer formation

1997 ◽  
Vol 326 (1) ◽  
pp. 31-38 ◽  
Author(s):  
John S. ELCE ◽  
Peter L. DAVIES ◽  
Carol HEGADORN ◽  
Donald H. MAURICE ◽  
J. Simon C. ARTHUR
Keyword(s):  
Catalytic Mechanism ◽  
Large Subunit ◽  
Small Subunit ◽  
Full Length ◽  
Deletion Mutants ◽  
Subunit Interactions ◽  
Calpain Activity ◽  
Specific Activities ◽  
The Stability ◽  
Histidine Tag

In order to study subunit interactions in calpain, the effects of small subunit truncations on m-calpain activity and heterodimer formation have been measured. It has been shown previously that active calpain is formed by co-expression of the large subunit (80 kDa) of rat m-calpain with a δ86 form (21 kDa) of the small subunit. cDNA for the full-length 270 amino acid (28.5 kDa) rat calpain small subunit has now been cloned, both with and without an N-terminal histidine tag (NHis10). The full-length small subunit constructs yielded active calpains on co-expression with the large subunit, and the small subunit was autolysed to 20 kDa on exposure of these calpains to Ca2+. A series of deletion mutants of the small subunit, NHis10-δ86,-δ99, -δ107, and -δ116, gave active heterodimeric calpains with unchanged specific activities, although in decreasing yield, and with a progressive decrease in stability. NHis10-δ125 formed a heterodimer which was inactive and unstable. Removal of 25 C-terminal residues from δ86, leaving residues 87–245, abolished both activity and heterodimer formation. The results show that: (a) generation of active m-calpain in Escherichia coli requires heterodimer formation; (b) small subunit residues between 94 and 116 contribute to the stability of the active heterodimer but do not directly affect the catalytic mechanism; (c) residues in the region 245–270 are essential for subunit binding. Finally, it was shown that an inactive mutant Cys105 → Ser-80k/δ86 calpain, used in order to preclude autolysis, did not dissociate in the presence of Ca2+, a result which does not support the proposal that Ca2+-induced dissociation is involved in calpain activation.

Bursaphelenchus sakishimanus n. sp. (Tylenchomorpha: Aphelenchoididae) isolated from a stag beetle, Dorcus titanus sakishimanus Nomura (Coleoptera: Lucanidae), on Ishigaki Island, Japan

Nematology ◽  
2015 ◽  
Vol 17 (5) ◽  
pp. 531-542 ◽  
Author(s):  
Natsumi Kanzaki ◽  
Kimiko Okabe ◽  
Youichi Kobori
Keyword(s):  
New Species ◽  
Ribosomal Rna ◽  
Large Subunit ◽  
Agar Plate ◽  
Small Subunit ◽  
Full Length ◽  
Ishigaki Island ◽  
Tropical America ◽  
Stag Beetles ◽  
Close Relatives

An undescribedBursaphelenchusspecies was isolated and cultured fromDorcus titanus sakishimanuscollected during a field survey of the insect-associated nematodes in subtropical Japan. The stag beetles were collected from Ishigaki Island, Okinawa, Japan, and dissected to examine their nematode associates. Then the dissected bodies were individually transferred to 2.0% agar plates, and nematode propagation on the plates was periodically examined. Nematodes were first recovered from the agar plate,i.e., the number of nematodes carried by the beetle was low, and infection was not confirmed during dissection. The new species was morphologically and phylogenetically (on a molecular basis) close toB. gerberae, which was isolated from the palm weevil,Rhynchophorus palmarum(Curculionidae), from tropical America, and to other weevil-associatedBursaphelenchusspecies. However, the new species can be distinguished from its close relatives by its typological characters,e.g., long and slender female tail and male spicule morphology as well as phylogenetic status inferred from the near-full-length of the small subunit (SSU) of the ribosomal RNA gene. The new species is described and illustrated herein asB. sakishimanusn. sp. and its molecular profiles, near-full-length SSU, and D2-D3 expansion segments of the large subunit of ribosomal RNA are described. This is the secondBursaphelenchusspecies associated with stag beetles (Lucanidae).

Acrostichus palmarum n. sp., a cryptic species separated from A. rhynchophori by molecular sequences and hybridisation tests

Nematology ◽  
2018 ◽  
Vol 20 (8) ◽  
pp. 751-768 ◽  
Author(s):  
Natsumi Kanzaki ◽  
Robin M. Giblin-Davis
Keyword(s):  
Large Subunit ◽  
Small Subunit ◽  
South Florida ◽  
Full Length ◽  
South American ◽  
Molecular Sequence ◽  
Sequence Profiles ◽  
Culture Code ◽  
Rhynchophorus Cruentatus ◽  
Independent Species

Summary A new Acrostichus species is described based upon molecular sequence profiles and hybridisation testing. The new species, A. palmarum n. sp., had been previously described as local isolates (strains) of A. rhynchophori, i.e., an isolate recovered from Rhynchophorus cruentatus from South Florida (culture code RGD193) was designated as the type strain of A. rhynchophori, and other Central and South American strains (RGD194-196), recovered from R. palmarum were considered as conspecific regional isolates. However, additional sequencing of ribosomal DNA loci (near full-length of small subunit, full length of internal transcribed spacer and D2-D3 expansion segments of large subunit) and partial mitochondrial cytochrome oxidase subunit I gene and hybridisation testing suggested the independent species status of RGD194-196. Furthermore, two strains of A. palmarum n. sp., RGD194 and RGD195, showed partial reproductive isolation from each other, i.e., the fecundity of F1 progeny was obviously low, suggesting that geographical isolation within a widely-distributed species is occurring.

Depletion and Reconstitution of Active E. Coli Ribosomes

1975 ◽  
Vol 33 ◽  
pp. 640-641
Author(s):  
M. Boublik ◽  
W. Hellmann ◽  
F. Jenkins
Keyword(s):  
Protein Biosynthesis ◽  
Large Subunit ◽  
High Resolution Electron Microscopy ◽  
Small Subunit ◽  
Structure And Function ◽  
Biologically Active ◽  
Biological Macromolecules ◽  
E Coli ◽  
Resolution Electron ◽  
And Function

Correlations between structure and function of biological macromolecules have been studied intensively for many years, mostly by indirect methods. High resolution electron microscopy is a unique tool which can provide such information directly by comparing the conformation of biopolymers in their biologically active and inactive state. We have correlated the structure and function of ribosomes, ribonucleoprotein particles which are the site of protein biosynthesis. 70S E. coli ribosomes, used in this experiment, are composed of two subunits - large (50S) and small (30S). The large subunit consists of 34 proteins and two different ribonucleic acid molecules. The small subunit contains 21 proteins and one RNA molecule. All proteins (with the exception of L7 and L12) are present in one copy per ribosome.This study deals with the changes in the fine structure of E. coli ribosomes depleted of proteins L7 and L12. These proteins are unique in many aspects.

Theileria parva ribosomal internal transcribed spacer sequences exhibit extensive polymorphism and mosaic evolution: application to the characterization of parasites from cattle and buffalo

Parasitology ◽  
1999 ◽  
Vol 118 (6) ◽  
pp. 541-551 ◽  
Author(s):  
N. E. COLLINS ◽  
B. A. ALLSOPP
Keyword(s):  
Genetic Recombination ◽  
Large Subunit ◽  
Small Subunit ◽  
Internal Transcribed Spacers ◽  
Rrna Genes ◽  
Gene Pools ◽  
Theileria Parva ◽  
Causative Agents ◽  
Internal Transcribed Spacer Sequences

We sequenced the rRNA genes and internal transcribed spacers (ITS) of several Theileria parva isolates in an attempt to distinguish between the causative agents of East coast fever and Corridor disease. The small subunit (SSU) and large subunit (LSU) rRNA genes from a cloned T. p. lawrencei parasite were sequenced; the former was identical to that of T. p. parva Muguga, and there were minor heterogeneities in the latter. The 5·8S gene sequences of 11 T. parva isolates were identical, but major differences were found in the ITS. Six characterization oligonucleotides were designed to hybridize within the variable ITS1 region; 93·5% of T. p. parva isolates examined were detected by probe TPP1 and 81·8% of T. p. lawrencei isolates were detected by TPL2 and/or TPL3a. There was no absolute distinction between T. p. parva and T. p. lawrencei and the former hybridized with fewer of the probes than did the latter. It therefore seems that a relatively homogenous subpopulation of T. parva has been selected in cattle from a more diverse gene pool in buffalo. The ITSs of both T. p. parva and T. p. lawrencei contained different combinations of identifiable sequence segments, resulting in a mosaic of segments in any one isolate, suggesting that the two populations undergo genetic recombination and that their gene pools are not completely separate.

scholarly journals Domains for protein-protein interactions at the N and C termini of the large subunit of bacteriophage lambda terminase.

Genetics ◽  
1988 ◽  
Vol 119 (3) ◽  
pp. 477-484
Author(s):  
W F Wu ◽  
S Christiansen ◽  
M Feiss
Keyword(s):  
Protein Interactions ◽  
Ternary Complex ◽  
Large Subunit ◽  
Small Subunit ◽  
Functional Domain ◽  
Binary Complex ◽  
Protein Protein Interactions ◽  
Related Phage ◽  
Carboxy Terminal ◽  
Lambda Dna

Abstract The large subunit of phage lambda terminase, gpA, the gene product of the phage A gene, interacts with the small subunit, gpNul, to form functional terminase. Terminase binds to lambda DNA at cosB to form a binary complex. The terminase:DNA complex binds a prohead to form a ternary complex. Ternary complex formation involves an interaction of the prohead with gpA. The amino terminus of gpA contains a functional domain for interaction with gpNul, and the carboxy-terminal 38 amino acids of gpA contain a functional domain for prohead binding. This information about the structure of gpA was obtained through the use of hybrid phages resulting from recombination between lambda and the related phage 21. lambda and 21 encode terminases that are analogous in structural organization and have ca. 60% sequence identity. In spite of these similarities, lambda and 21 terminases differ in specificity for DNA binding, subunit assembly, and prohead binding. A lambda-21 hybrid phage produces a terminase in which one of the subunits is chimeric and had recombinant specificities. In the work reported here; a new hybrid, lambda-21 hybrid 67, is characterized. lambda-21 hybrid 67 is the result of a crossover between lambda and 21 in the large subunit genes, such that the DNA from the left chromosome end is from 21, including cosB phi 21, the 1 gene, and the first 48 codons for the 2 gene. The rest of the hybrid 67 chromosome is lambda DNA, including 593 codons of the A gene. The chimeric gp2/A of hybrid 67 binds gp1 to form functional terminase.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals The Evolution of Life Modes in Stictidaceae, with Three Novel Taxa

2021 ◽  
Vol 7 (2) ◽  
pp. 105
Author(s):  
Vinodhini Thiyagaraja ◽  
Robert Lücking ◽  
Damien Ertz ◽  
Samantha C. Karunarathna ◽  
Dhanushka N. Wanasinghe ◽  
...  
Keyword(s):  
New Species ◽  
Large Scale ◽  
Sequence Data ◽  
Large Subunit ◽  
Monte Carlo Sampling ◽  
Small Subunit ◽  
Sensu Stricto ◽  
Internal Transcribed Spacers ◽  
Character State ◽  
Phenotypic Data

Ostropales sensu lato is a large group comprising both lichenized and non-lichenized fungi, with several lineages expressing optional lichenization where individuals of the same fungal species exhibit either saprotrophic or lichenized lifestyles depending on the substrate (bark or wood). Greatly variable phenotypic characteristics and large-scale phylogenies have led to frequent changes in the taxonomic circumscription of this order. Ostropales sensu lato is currently split into Graphidales, Gyalectales, Odontotrematales, Ostropales sensu stricto, and Thelenellales. Ostropales sensu stricto is now confined to the family Stictidaceae, which includes a large number of species that are poorly known, since they usually have small fruiting bodies that are rarely collected, and thus, their taxonomy remains partly unresolved. Here, we introduce a new genus Ostropomyces to accommodate a novel lineage related to Ostropa, which is composed of two new species, as well as a new species of Sphaeropezia, S. shangrilaensis. Maximum likelihood and Bayesian inference analyses of mitochondrial small subunit spacers (mtSSU), large subunit nuclear rDNA (LSU), and internal transcribed spacers (ITS) sequence data, together with phenotypic data documented by detailed morphological and anatomical analyses, support the taxonomic affinity of the new taxa in Stictidaceae. Ancestral character state analysis did not resolve the ancestral nutritional status of Stictidaceae with confidence using Bayes traits, but a saprotrophic ancestor was indicated as most likely in a Bayesian binary Markov Chain Monte Carlo sampling (MCMC) approach. Frequent switching in nutritional modes between lineages suggests that lifestyle transition played an important role in the evolution of this family.

Morphometrics and molecular analysis ofOzolaimus linstowin. sp. (Oxyuroidea: Pharyngodonidae) from the green lizardIguana iguana

2015 ◽  
Vol 90 (2) ◽  
pp. 186-198 ◽  
Author(s):  
S.V. Malysheva
Keyword(s):  
Body Size ◽  
Large Subunit ◽  
Buccal Capsule ◽  
Small Subunit ◽  
Present Species ◽  
Lsu Rdna ◽  
Adult Males ◽  
Spicule Length ◽  
Males And Females ◽  
Characteristic Features

AbstractOzolaimus linstowin. sp. is described from the large intestine ofIguana iguanaLinnaeus, 1758 from Mexico. The present species can be easily distinguished fromO. megatyphlonandO. cirratusby the presence of a long and slender pharynx not divided into sections, more similar to the remaining two species,O. monhysteraandO. ctenosauri. Ozolaimus linstowin. sp. can be differentiated fromO. monhysteraby the shorter spicule length and smaller body size of both males and females. Males ofO. linstowin. sp. are morphologically close to those ofO. ctenosauri, but females possess a markedly smaller body size and differ in the organization of the oral cuticular armature. Adult males ofO. linstowin. sp. bear some characteristic features of the J3 juvenile morphology in terms of the cuticular organization of the oral and buccal capsule. Phylogenetic analysis ofO.linstowin. sp. using partial small subunit (SSU) and D2–D3 large subunit (LSU) rDNA shows relationships with several Oxyuridae genera.

scholarly journals Molecular Diversity of the Syndinean Genus Euduboscquella Based on Single-Cell PCR Analysis

2011 ◽  
Vol 78 (2) ◽  
pp. 334-345 ◽  
Author(s):  
Tsvetan R. Bachvaroff ◽  
Sunju Kim ◽  
Laure Guillou ◽  
Charles F. Delwiche ◽  
D. Wayne Coats
Keyword(s):  
Large Subunit ◽  
Small Subunit ◽  
Base Change ◽  
Pcr Analysis ◽  
Ssu Rrna ◽  
Compensatory Base Change ◽  
Content Type ◽  
Single Cell Pcr ◽  
Group I ◽  
Lsu Rrna

ABSTRACTThe genusEuduboscquellais one of a few described genera within the syndinean dinoflagellates, an enigmatic lineage with abundant diversity in marine environmental clone libraries based on small subunit (SSU) rRNA. The region composed of the SSU through to the partial large subunit (LSU) rRNA was determined from 40 individual tintinnid ciliate loricae infected withEuduboscquellasampled from eight surface water sites in the Northern Hemisphere, producing seven distinct SSU sequences. The corresponding host SSU rRNA region was also amplified from eight host species. The SSU tree ofEuduboscquellaand syndinean group I sequences from environmental clones had seven well-supported clades and one poorly supported clade across data sets from 57 to 692 total sequences. The genusEuduboscquellaconsistently formed a supported monophyletic clade within a single subclade of group I sequences. For most parasites with identical SSU sequences, the more variable internal transcribed spacer (ITS) to LSU rRNA regions were polymorphic at 3 to 10 sites. However, inE. cachonithere was variation between ITS to LSU copies at up to 20 sites within an individual, while in a parasite ofTintinnopsisspp., variation between different individuals ranged up to 19 polymorphic sites. However, applying the compensatory base change model to the ITS2 sequences suggested no compensatory changes within or between individuals with the same SSU sequence, while one to four compensatory changes between individuals with similar but not identical SSU sequences were found. Comparisons between host and parasite phylogenies do not suggest a simple pattern of host or parasite specificity.

scholarly journals Investigation of in Vivo Roles of the C-terminal Tails of the Small Subunit (ββ′) of Saccharomyces cerevisiae Ribonucleotide Reductase

2013 ◽  
Vol 288 (20) ◽  
pp. 13951-13959 ◽  
Author(s):  
Yan Zhang ◽  
Xiuxiang An ◽  
JoAnne Stubbe ◽  
Mingxia Huang
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ribonucleotide Reductase ◽  
Large Subunit ◽  
Small Subunit ◽  
Cluster Assembly ◽  
E Coli ◽  
Viability Assay ◽  
Docking Model

The small subunit (β2) of class Ia ribonucleotide reductase (RNR) houses a diferric tyrosyl cofactor (Fe2III-Y•) that initiates nucleotide reduction in the large subunit (α2) via a long range radical transfer (RT) pathway in the holo-(α2)m(β2)n complex. The C-terminal tails of β2 are predominantly responsible for interaction with α2, with a conserved tyrosine residue in the tail (Tyr356 in Escherichia coli NrdB) proposed to participate in cofactor assembly/maintenance and in RT. In the absence of structure of any holo-RNR, the role of the β tail in cluster assembly/maintenance and its predisposition within the holo-complex have remained unknown. In this study, we have taken advantage of the unusual heterodimeric nature of the Saccharomyces cerevisiae RNR small subunit (ββ′), of which only β contains a cofactor, to address both of these issues. We demonstrate that neither β-Tyr376 nor β′-Tyr323 (Tyr356 equivalent in NrdB) is required for cofactor assembly in vivo, in contrast to the previously proposed mechanism for E. coli cofactor maintenance and assembly in vitro. Furthermore, studies with reconstituted-ββ′ and an in vivo viability assay show that β-Tyr376 is essential for RT, whereas Tyr323 in β′ is not. Although the C-terminal tail of β′ is dispensable for cofactor formation and RT, it is essential for interactions with β and α to form the active holo-RNR. Together the results provide the first evidence of a directed orientation of the β and β′ C-terminal tails relative to α within the holoenzyme consistent with a docking model of the two subunits and argue against RT across the β β′ interface.

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