Characterization of a Single-Copy Arabidopsis Gene Encoding a Protein Showing Limited Similarity to the N-terminus of the Mammalian Clathrin-Assembly Protein AP180

RNA components have been identified in preparations of RNase P from a number of eucaryotic sources, but final proof that these RNAs are true RNase P subunits has been elusive because the eucaryotic RNAs, unlike the procaryotic RNase P ribozymes, have not been shown to have catalytic activity in the absence of protein. We previously identified such an RNA component in Saccharomyces cerevisiae nuclear RNase P preparations and have now characterized the corresponding, chromosomal gene, called RPR1 (RNase P ribonucleoprotein 1). Gene disruption experiments showed RPR1 to be single copy and essential. Characterization of the gene region located RPR1 600 bp downstream of the URA3 coding region on chromosome V. We have sequenced 400 bp upstream and 550 bp downstream of the region encoding the major 369-nucleotide RPR1 RNA. The presence of less abundant, potential precursor RNAs with an extra 84 nucleotides of 5' leader and up to 30 nucleotides of 3' trailing sequences suggests that the primary RPR1 transcript is subjected to multiple processing steps to obtain the 369-nucleotide form. Complementation of RPR1-disrupted haploids with one variant of RPR1 gave a slow-growth and temperature-sensitive phenotype. This strain accumulates tRNA precursors that lack the 5' end maturation performed by RNase P, providing direct evidence that RPR1 RNA is an essential component of this enzyme.

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Molecular cloning and characterization of the structural gene for the major iron-regulated protein expressed by Neisseria gonorrhoeae.

Journal of Experimental Medicine ◽

10.1084/jem.171.5.1535 ◽

1990 ◽

Vol 171 (5) ◽

pp. 1535-1546 ◽

Cited By ~ 35

Author(s):

S A Berish ◽

T A Mietzner ◽

L W Mayer ◽

C A Genco ◽

B P Holloway ◽

...

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Neisseria Gonorrhoeae ◽

Biochemical Characterization ◽

Iron Acquisition ◽

Regulatory Region ◽

Single Copy ◽

Oligonucleotide Probes ◽

Gene Encoding

This report describes the cloning and sequencing of the major iron-regulated protein (termed Fbp) of Neisseria gonorrhoeae strain F62. Attempts to identify recombinants expressing the Fbp using specific antibody proved unsuccessful. Therefore, an alternative cloning strategy using oligonucleotide probes derived from NH2-terminal and tryptic fragments of this protein was used to identify short fragments of the gene. Using this methodology, the gene encoding the precursor of Fbp was cloned on three separate overlapping fragments and sequenced, and the amino acid sequence was deduced. These data were unambiguously confirmed by the known NH2-terminal amino acid sequence and were supported by the sequences from tryptic fragments that lie outside of this region. Using oligonucleotide probes, we were unable to obtain clones encoding the potential regulatory region of this protein. Therefore, the technique of inverse polymerase chain reaction was used to amplify a fragment containing an additional 200 bp. This fragment was cloned and sequenced and found to contain a consensus ribosome binding site and potential -10 and -35 sequences. Hybridization analysis of genomic DNA from gonococcal strain F62 indicated that only a single copy of the Fbp gene exists per genome. These results complement the biochemical characterization of the Fbp expressed by gonococci and further suggest that it has a role in iron-acquisition.

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Molecular characterization of a gene encoding N-myristoyl transferase (NMT) from Triticum aestivum (bread wheat)

Genome ◽

10.1139/g04-074 ◽

2004 ◽

Vol 47 (6) ◽

pp. 1036-1042 ◽

Cited By ~ 8

Author(s):

Tim Dumonceaux ◽

Raju V.S Rajala ◽

Rajendra Sharma ◽

Gopalan Selvaraj ◽

Raju Datla

Keyword(s):

Triticum Aestivum ◽

Protein Modification ◽

Wheat Plant ◽

Single Copy ◽

Gene Encoding ◽

Growth And Survival ◽

Reading Frame ◽

Eukaryotic Proteins ◽

Taxonomic Groups

Myristoyl-CoA:protein N-myristoyl transferase (NMT; EC 2.3.1.97) acylates the Gly residue abutting the N-terminal Met with a myristic acid following the removal of the Met residue in certain eukaryotic proteins, and in some cases myristoylation is essential to cell growth and survival. We report the cloning of a full-length cDNA encoding NMT from Triticum aestivum (TaNMT). The cDNA included a predicted open reading frame of 1317 nucleotides, which encoded a predicted protein of 438 amino acids containing all of the residues that are important for NMT activity. The TaNMT amino acid and nucleotide sequences were compared with NMTs from 14 other species encompassing a wide array of taxonomic groups. Among the experimentally validated NMTs, TaNMT was most similar to that of Arabidopsis thaliana. Southern blot analysis of wheat genomic DNA showed that TaNMT is encoded by a single copy gene, with one copy per haploid genome. We expressed TaNMT in Escherichia coli cells and determined that the recombinant protein possessed NMT activity, catalyzing the N-myristoylation of peptides from known or putatively myristoylated proteins from plants and animals without a strong preference for the plant peptides. TaNMT is the second experimentally validated plant NMT sequence and the first from a monocotyledonous species.Key words: N-myristoyl transferase, myristoylation, protein modification, wheat, plant development.

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The N Terminus of Rotavirus VP2 Is Necessary for Encapsidation of VP1 and VP3

Journal of Virology ◽

10.1128/jvi.72.1.201-208.1998 ◽

1998 ◽

Vol 72 (1) ◽

pp. 201-208 ◽

Cited By ~ 54

Author(s):

Carl Q.-Y. Zeng ◽

Mary K. Estes ◽

Annie Charpilienne ◽

Jean Cohen

Keyword(s):

Inner Core ◽

Major Capsid Protein ◽

Core Layer ◽

Structural Protein ◽

Gene Encoding ◽

Double Stranded Rna ◽

N Terminus ◽

Far Western Blot

ABSTRACT The innermost core of rotavirus is composed of VP2, which forms a protein layer that surrounds the two minor proteins VP1 and VP3, and the genome of 11 segments of double-stranded RNA. This inner core layer surrounded by VP6, the major capsid protein, constitutes double-layered particles that are transcriptionally active. Each gene encoding a structural protein of double-layered particles has been cloned into baculovirus recombinants and expressed in insect cells. Previously, we showed that coexpression of different combinations of the structural proteins of rotavirus double-layered particles results in the formation of virus-like particles (VLPs), and each VLP containing VP1, the presumed RNA-dependent RNA polymerase, possesses replicase activity as assayed in an in vitro template-dependent assay system (C. Q.-Y. Zeng, M. J. Wentz, J. Cohen, M. E. Estes, and R. F. Ramig, J. Virol. 70:2736–2742, 1996). This work reports construction and characterization of VLPs containing a truncated VP2 (VPΔ2, containing amino acids [aa] Met-93 to 880). Expression of VPΔ2 alone resulted in the formation of single-layered Δ2-VLPs. Coexpression of VPΔ2 with VP6 produced double-layered Δ2/6-VLPs. VLPs formed by coexpression of VPΔ2 and VP1 or VP3, or both VP1 and VP3, resulted in the formation of VLPs lacking both VP1 and VP3. The presence of VP6 with VPΔ2 did not result in encapsidation of VP1 and VP3. To determine the domain of VP2 required for binding VP1, far-Western blot analyses using a series of truncated VP2 constructs were performed to test their ability to bind VP1. These analyses showed that (i) full-length VP2 (aa 1 to 880) binds to VP1, (ii) any N-terminal truncation lacking aa 1 to 25 fails to bind VP1, and (iii) a C-terminal 296-aa truncated VP2 construct (aa 1 to 583) maintains the ability to bind VP1. These analyses indicate that the N terminus of rotavirus VP2 is necessary for the encapsidation of VP1 and VP3.

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Characterization of RPR1, an essential gene encoding the RNA component of Saccharomyces cerevisiae nuclear RNase P

Molecular and Cellular Biology ◽

10.1128/mcb.11.2.721-730.1991 ◽

1991 ◽

Vol 11 (2) ◽

pp. 721-730 ◽

Cited By ~ 1

Author(s):

J Y Lee ◽

C E Rohlman ◽

L A Molony ◽

D R Engelke

Keyword(s):

Saccharomyces Cerevisiae ◽

Essential Gene ◽

Rnase P ◽

Single Copy ◽

Temperature Sensitive ◽

Coding Region ◽

Gene Encoding ◽

Processing Steps ◽

Potential Precursor

RNA components have been identified in preparations of RNase P from a number of eucaryotic sources, but final proof that these RNAs are true RNase P subunits has been elusive because the eucaryotic RNAs, unlike the procaryotic RNase P ribozymes, have not been shown to have catalytic activity in the absence of protein. We previously identified such an RNA component in Saccharomyces cerevisiae nuclear RNase P preparations and have now characterized the corresponding, chromosomal gene, called RPR1 (RNase P ribonucleoprotein 1). Gene disruption experiments showed RPR1 to be single copy and essential. Characterization of the gene region located RPR1 600 bp downstream of the URA3 coding region on chromosome V. We have sequenced 400 bp upstream and 550 bp downstream of the region encoding the major 369-nucleotide RPR1 RNA. The presence of less abundant, potential precursor RNAs with an extra 84 nucleotides of 5' leader and up to 30 nucleotides of 3' trailing sequences suggests that the primary RPR1 transcript is subjected to multiple processing steps to obtain the 369-nucleotide form. Complementation of RPR1-disrupted haploids with one variant of RPR1 gave a slow-growth and temperature-sensitive phenotype. This strain accumulates tRNA precursors that lack the 5' end maturation performed by RNase P, providing direct evidence that RPR1 RNA is an essential component of this enzyme.

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Characterization of emb, a Gene Encoding the Major Adhesin of Streptococcus defectivus

Infection and Immunity ◽

10.1128/iai.67.1.50-56.1999 ◽

1999 ◽

Vol 67 (1) ◽

pp. 50-56 ◽

Cited By ~ 11

Author(s):

Riccardo Manganelli ◽

Ivo van de Rijn

Keyword(s):

Amino Acids ◽

Molecular Weight ◽

Extracellular Matrix ◽

Coiled Coil ◽

Surface Protein ◽

Gene Encoding ◽

New Family ◽

N Terminus ◽

Viridans Group Streptococci

ABSTRACT Streptococcus defectivus is one of the nutritionally variant streptococci, a class of viridans group streptococci first isolated from patients with endocarditis and otitis media. In previous studies, NVS-47, a clinical isolate of S. defectivus, was shown to bind to the extracellular matrix. A high-molecular-weight surface protein was identified and proposed to be responsible for mediating this binding. In the present study, the gene encoding this protein was identified by transposon mutagenesis and characterized. The gene (emb) was found to be larger than 14 kb and was partially sequenced. It encodes a protein containing at least 50 repeats of 77 amino acids predicted to assume an alternating coiled-coil conformation. The domain responsible for extracellular matrix binding was mapped to the N terminus of the protein. From sequence analysis, Emb is proposed to be the prototype of a new family of streptococcal fibrillar proteins.

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Molecular Characterization of an Arabidopsis Gene Encoding a Phospholipid-Specific Inositol Polyphosphate 5-Phosphatase

PLANT PHYSIOLOGY ◽

10.1104/pp.104.040253 ◽

2004 ◽

Vol 135 (2) ◽

pp. 938-946 ◽

Cited By ~ 32

Author(s):

Mustafa E. Ercetin ◽

Glenda E. Gillaspy

Keyword(s):

Molecular Characterization ◽

Inositol Polyphosphate ◽

Gene Encoding ◽

Arabidopsis Gene

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Characterization of a single copy gene encoding ferredoxin I from pea.

The Plant Cell ◽

10.1105/tpc.1.7.681 ◽

1989 ◽

Vol 1 (7) ◽

pp. 681-690 ◽

Cited By ~ 22

Author(s):

R C Elliott ◽

T J Pedersen ◽

B Fristensky ◽

M J White ◽

L F Dickey ◽

...

Keyword(s):

Single Copy ◽

Single Copy Gene ◽

Gene Encoding ◽

Copy Gene ◽

Ferredoxin I

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Characterization of the gene encoding glyoxalase II from Leishmania donovani: a potential target for anti-parasite drugs

Biochemical Journal ◽

10.1042/bj20050948 ◽

2005 ◽

Vol 393 (1) ◽

pp. 227-234 ◽

Cited By ~ 30

Author(s):

Prasad K. Padmanabhan ◽

Angana Mukherjee ◽

Rentala Madhubala

Keyword(s):

Substrate Specificity ◽

Leishmania Donovani ◽

Affinity Purification ◽

Single Copy ◽

Cellular Damage ◽

Gene Encoding ◽

Structure Based Drug Design ◽

Calculated Molecular Mass ◽

Glyoxalase Ii

The glyoxalase system is a ubiquitous detoxification pathway that protects against cellular damage caused by highly reactive oxoaldehydes such as methylglyoxal which is mainly formed as a by-product of glycolysis. The gene encoding GLOII (glyoxalase II) has been cloned from Leishmania donovani, a protozoan parasite that causes visceral leishmaniasis. DNA sequence analysis revealed an ORF (open reading frame) of ∼888 bp that encodes a putative 295-amino-acid protein with a calculated molecular mass of 32.5 kDa and a predicted pI of 6.0. The sequence identity between human GLOII and LdGLOII (L. donovani GLOII) is only 35%. The ORF is a single-copy gene on a 0.6-Mb chromosome. A ∼38 kDa protein was obtained by heterologous expression of LdGLOII in Escherichia coli, and homogeneous enzyme was obtained after affinity purification. Recombinant L. donovani GLOII showed a marked substrate specificity for trypanothione hemithioacetal over glutathione hemithioacetal. Antiserum against recombinant LdGLOII protein could detect a band of anticipated size ∼32 kDa in promastigote extracts. By overexpressing the GLOII gene in Leishmania donovani using Leishmania expression vector pspαhygroα, we detected elevated expression of GLOII RNA and protein. Overexpression of the GLOII gene will facilitate studies of gene function and its relevance as a chemotherapeutic target. This is the first report on the molecular characterization of glyoxalase II from Leishmania spp. The difference in the substrate specificity of the human and Leishmania donovani glyoxalase II enzyme could be exploited for structure-based drug design of selective inhibitors against the parasite.

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