Page-McCaw PS et al. (1999) RNA "PUF60: a novel U2AF65-related splicing activity."

Page-McCaw PS, Sharp PA, Amonlirdviman K

[RNA, 1999]

We have identified a new pyrimidine-tract binding factor, PUF, that is required, together with U2AF, for efficient reconstitution of RNA splicing in vitro. The activity has been purified and consists of two proteins, PUF60 and the previously described splicing factor p54. p54 and PUF60 form a stable complex in vitro when cotranslated in a reaction mixture. PUF activity, in conjunction with U2AF, facilitates the association of U2 snRNP with the pre-mRNA. This reaction is dependent upon the presence of the large subunit of U2AF, U2AF65, but not the small subunit U2AF35. PUF60 is homologous to both U2AF65 and the yeast splicing factor Mud2p. The C-terminal domain of PUF60, the PUMP domain, is distantly related to the RNA-recognition motif domain, and is probably important in protein-protein interactions.

Inoue T et al. (2007) Vet Parasitol "Partial characterization of the antigen recognized by a monoclonal antibody to Ascaris suum ovary extracts."

Takashima M, Watanabe T, Inoue T, Murakami S

[Vet Parasitol, 2007]

A monoclonal antibody produced against ovary extracts from the worm Ascaris suum showed immunoreactivity against granules in the rachis and oocytes, the inner layer of the eggshell and the middle layer of some egg, but not against either ovary wall or uterus wall. Furthermore, the same antigens were detected on the body surface of migrated larva in guinea pig lung, whereas none were detected in adult male worm or adult female worm, except for the female reproductive organs. The ovary extracts were passed through an affinity column and the eluted fractions analyzed by SDS-PAGE, Western blotting and native-PAGE. Western blotting after SDS-PAGE detected chemiluminescence primarily as three bands of about 70, 78 and 90kDa. However, Western blotting after native-PAGE of the partially purified ovary extracts demonstrated only one band at a position of about 230kDa. LC-nanoESI-MS/MS analysis of protein band gel slices from silver-stained SDS-PAGE revealed one peptide sequence "ILVGLIGTNR", that matched only the hypothetical protein F14D2.8 of Caenorhabditis elegans (gi/7499081).

Hokii Y et al. (2006) Gene "Twelve novel C. elegans RNA candidates isolated by two-dimensional polyacrylamide gel electrophoresis."

Hokii Y, Ogasawara T, Taneda A, Arai R, Kubo A, Ushida C, Nogi Y, Muto A

[Gene, 2006]

C. elegans small RNAs (<50 nt) were separated by two-dimensional gel electrophoresis (2D-PAGE). cDNAs were prepared from the RNAs extracted from randomly chosen 2D-PAGE spots. Although many cDNA sequences corresponded to parts of known RNAs, twelve novel small RNA candidates were identified: eleven from 2D-PAGE spots of the mixed-stage worm RNA preparation and one from those of the embryonic RNA preparation. These are encoded in the intergenic regions, in the introns of protein-coding genes, in the anti-sense strand of protein-coding sequences and repetitive sequence regions of the genome. None of them showed a characteristic structure of miRNAs, suggesting that they are candidates of other or new classes of RNAs.

van den Ecker D et al. (2010) Anal Biochem "Blue native electrophoresis to study mitochondrial complex I in C. elegans."

van den Brand MA, Nijtmans LG, Distelmaier F, van den Ecker D, Bossinger O, Mayatepek E

[Anal Biochem, 2010]

Blue native polyacrylamide gel electrophoresis (BN-PAGE) is an essential tool for investigating mitochondrial respiratory chain complexes. However, with current BN-PAGE protocols for Caenorhabditis elegans (C. elegans), large worm amounts and high quantities of mitochondrial protein are required to yield clear results. Here, we present an efficient approach to isolate mitochondrial complex I (NADH:ubiquinone oxidoreductase) from C. elegans, grown on agar plates. We demonstrate that considerably lower amounts of mitochondrial protein are sufficient to isolate complex I and to display clear in-gel activity results. Moreover, we present the first complex I assembly profile for C. elegans, obtained by two-dimensional BN/SDS-PAGE.

Bargmann C (2000) Neurobiol Dis "Simple organisms."

Bargmann C

[Neurobiol Dis, 2000]

How are researchers now able to study the complex human genome with such insight and accuracy? The answer lies in nearly a century of research with very simple organisms: the fruit fly, Drosophila melanogaster, which is about 1/8 in. in size, and the roundworm, Caenorhabditis elegans, about the size of a comma on this page.

Kaushal NA et al. (1982) J Immunol "Identification and characterization of excretory-secretory products of Brugia malayi, adult filarial parasites."

Kaushal NA, Ottesen EA, Nash TE, Hussain R

[J Immunol, 1982]

Although E-S antigens may be particularly important for both the pathogenesis and immunodiagnosis of helminth infections, little is known about the immunochemistry or functional roles in human filarial infections. In the present paper, we have done some initial identification and characterization of E-S products of adult Brugia malayi by employing a combination of sensitive biochemical and immunochemical techniques. E-S products, collected by incubating B. malayi adults in vitro in a defined protein-free medium, were radiolabeled with 125I. SDS-polyacrylamide gel electrophoresis (PAGE) and autoradiography of labeled E-S products revealed 11 protein bands in the m.w. range of 10,000 to 70,000. Comparison of radiolabeled E-S products and adult somatic antigen (B.m.A) in SDS-PAGE indicated many common bands, and crossed immunoelectrophoresis and competitive Staph-A RIA confirmed the presence of most E-S antigens in B.m.A. Of the 11 E-S bands, two appeared to be derived from the surface of the adult worms and microfilariae as shown by SDS-PAGE and autoradiography of lodogen surface-labeled parasites; the presence of two host proteins in E-S was detected by crossed-line immunoelectrophoresis. The E-S antigens were highly immunogenic when tested both with rabbit antiserum raised against B.m.A and with a serum pool of patients with natural filarial infection.

Kaji H et al. (2000) Electrophoresis "Profiling of Caenorhabditis elegans proteins using two-dimensional gel electrophoresis and matrix assisted laser desorption/ionization-time of flight-mass spectrometry."

Mawuenyega KG, Isobe T, Kaji H, Taoka M, Wakamiya A, Tsuji T

[Electrophoresis, 2000]

The nematode Caenorhabditis elegans (C. elegans) is the first animal whose whole 97 Mb genome sequence, encoding ca. 19000 open reading frames (ORF's), has been essentially determined. We tried to establish a 2-DE map of the nematode proteome by means of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). A soluble protein fraction of mixed stages of the worm, wild-type strain N2, was applied to 2-D PAGE. After Coomassie Brilliant Blue (CBB) staining, 1200 spots were detected and 140 major spots were excised from the gel and subjected to in-gel digestion with Achromobacter protease I (lysyl endopeptidase). Resulting peptides were analyzed by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) followed by peptide mass fingerprinting for protein identification. With this approach we have obtained a two-dimensional electrophoresis (2-DE) protein map in which 69 spots were localized as landmarks for comparison of expression profiles to elucidate the basis of various biological events.

Russo, Anna E. et al. (2021) MicroPubl Biol

Nelson, Christian R., Bhalla, Needhi, Russo, Anna E.

[MicroPubl Biol, 2021]

Haploid sex cells such as sperm and eggs depend on a specialized form of cell division called meiosis. Throughout prophase of meiosis, homologous chromosomes pair, synapse, and recombine to form a physical link between them called a chiasma. Chiasmata are essential for proper segregation (Page et al. 2003). Defects in meiotic recombination can lead to gametes that contain an incorrect number of chromosomes (aneuploidy), which can cause infertility, miscarriages, and genetic disorders such as Down Syndrome. Therefore, identifying how recombination is regulated is crucial for understanding multiple aspects of human reproductive health.

Hammerberg B et al. (1989) J Immunol "Protective immunity against Brugia malayi infective larvae in mice. II. Induction by a T cell-dependent antigen isolated by monoclonal antibody affinity chromatography and SDS-PAGE."

Nogami S, Hammerberg B, Hayashi Y, Tanaka H, Nakagaki K

[J Immunol, 1989]

A mAb directed against filarial worm secretory/excretory product and reactive with Brugia malayi larval worm surface was used in conjunction with preparative SDS-PAGE to isolate protective Ag from extracts of adult B. malayi. The IgM mAb OVH bound to a repeating carbohydrate epitope present in adult, infective, and fourth stage larvae and microfilariae of B. malayi, and on the surface of fourth stage larvae. Ag bearing this epitope were also present in the sera of hosts infected with a variety of helminths, including Brugia, Onchocerca, Dirofilaria, and Paragonimus. Affinity chromatography of SDS extract of adult Brugia, using mAb OVH immobilized on agarose beads, isolated several Ag that separated into multiple protein staining bands on SDS-PAGE. In comparing SDS-PAGE-fractionated Ag from the crude SDS extract with fractionated mAb OVH-isolated Ag for the ability to protect BALB/c mice from challenge with B. malayi-infective larvae, it was found that of the mAb OVH-isolated Ag only those at a molecular mass of 26 to 32 kDa were protective while the original SDS extract yielded protective Ag at the following molecular mass: greater than 200, 170 to 200, 40 to 44, 33 to 36, 23 to 28, 20 to 22, and 17 to 19 kDa. Although Ag isolated by mAb OVH were highly protective, they failed to induce high antibody levels against the immunogen or SDS extracts compared to crude SDS extract immunized mouse sera, as determined by immunoblot and ELISA. Transfer of nylon wool non-adherent T cells from BALB/c mice immunized with the 26- to 28-kDa fraction of mAb OVH-isolated Ag to naive mice just before challenge with infective larvae of B. malayi resulted in a 70% reduction in larvae recovered 14 days after challenge.

Johnson TE et al. (1985) Mech Ageing Dev "Programmed aging or error catastrophe? An examination by two-dimensional polyacrylamide gel electrophoresis."

McCaffrey G, Johnson TE

[Mech Ageing Dev, 1985]

We have examined newly synthesized proteins in the young adult and in older populations of the nematode Caenorhabditis elegans using two- dimensional polyacrylamide gel electrophoresis (2D PAGE). A temperature-sensitive mutant strain, DH26, with a mean life span of about 15 days, under our conditions, was used to block progeny development. Nematodes of several different ages were pulse-labeled for 5 h, in vivo, with 35S-labeled E. coli, A subsequent 30-min chase with unlabeled E. coli served to rid the worms of endogenous labeled E. coli proteins. We resolve 700 or more proteins by 2D PAGE polyacrylamide gel electrophoresis of extracts of young nematodes. The patterns of these proteins are highly reproducible in comparisons of independent repeats of identical experiments. No new major proteins are synthesized at any time during the adult phase (4-22 days) nor are any of the most abundant proteins not made during this period. At our level of detectability (estimated as a satellite spot containing 4% of the amount of label in a major spot) we see no misincorporation of radioactive amino acids into newly synthesized proteins. These data are inconsistent with predictions by any one of several, so called, "error catastrophe" models of senescence and also show that modulation of the highest abundancy classes of