The autoantigen p43 is a nuclear protein initially identified with autoantibodies from dogs with a lupus-like syndrome. Here we show that p43 is an RNA-binding protein, and identify it as hnRNP G, a previously described component of heterogeneous nuclear ribonucleoprotein complexes. We demonstrate that p43/hnRNP G is glycosylated, and identify the modification as O-linked N-acetylglucosamine. A full-length cDNA clone for hnRNP G has been isolated and sequenced, and the predicted amino acid sequence for hnRNP G shows that it contains one RNP-consensus RNA binding domain (RBD) at the amino terminus and a carboxyl domain rich in serines, arginines and glycines. The RBD of human hnRNP G shows striking similarities with the RBDs of several plant RNA-binding proteins.
We used mAb A33/22, which recognizes a nuclear protein on the loops of amphibian lampbrush chromosomes, to select cDNA clone PwA33 from an expression library of the newt Pleurodeles waltl. A myc‐tagged transcript of clone PwA33 was injected into Pleurodeles oocytes. The translation product localized in the germinal vesicle (GV) and was distributed on the lampbrush loops in a pattern identical to that of the endogenous protein. PwA33 encodes a 71 kDa protein with three distinct domains: a region rich in Cys/His residues that may form zinc fingers, a coiled‐coil domain with potential for dimerization and a third ‘rfp‐like’ domain that is shared by several other nuclear proteins. The putative zinc fingers and the coiled‐coil domain resemble features in known nucleic acid‐binding regulatory proteins. These structures, coupled with a distinctive pattern of expression in embryonic tissues, suggest that A33 may function as a regulatory protein during early development. It is unlikely that the large store of A33 in the GV is bound to DNA. Instead, its association with the nascent transcripts on the lampbrush chromosome loops suggests a role in pre‐mRNA synthesis or processing.
Germinal vesicles of oocytes from Pleurodeles waltlii were used for immunization of BALB/c mice to obtain hybridomas secreting monoclonal antibodies. The hybridomas were screened for reactivity of their antibodies against lampbrush chromosomes of oocytes, as revealed by indirect immunostaining. Antibodies labelling the lampbrush chromosomes were also tested on histological sections of oocytes, embryos, and larvae of Pleurodeles. Characterization of the antigens was accomplished through immunoblotting of two-dimensional electrophoretic gels of germinal vesicle proteins. The ten monoclonal antibodies giving a positive reaction were classed into five groups. Group 1, exemplified by antibody A33, recognizes all the lampbrush chromosome transcribing sites (loops). Moreover, it differentially labels the cell nuclei during embryonic and larval development. Group 2, antibody B71, also stains all the loops of the lampbrush chromosomes, but does not react with cell nuclei of embryos and larvae. Group 3, antibody A1, labels specific loops, some of which are heterozygous in the strain of P. waltlii used. These heterozygosities have allowed us to localize and to characterize a chromosomal segment on bivalent IV which is heteromorphic in the two partners of the bivalent. We suggest that this heteromorphism represents a morphological distinction between Z and W heterochromosomes. Moreover, this antibody reacts with only one transcription unit along a loop that contains several units. Group 4, antibody B24, stains the only two structures in the lampbrush chromosomes of P. waltlii that do not have a loop organization, the mass "M" and the spheres. Group 5, antibody A35, reacts with the chromomeres. The antigens corresponding to antibodies A33 and B24 have been identified as proteins, which have apparent molecular weights of 80 and 104 kilodaltons, respectively. They correspond to proteins abundant in the germinal vesicles. All the antibodies described here cross-react with the lampbrush chromosomes of five other species of Urodeles.
The method of spreading transcription complexes has been applied to amphibian oocytes of Pleurodeles genus. Complexes of nucleolar origin show a regular and homogeneous organization similar to that described in other materials. The observations add to the interpretation as an amplification of nucleolar DNA and a redundancy of ribosomal cistrons in the two species studied. -- On the other hand, complexes of chromosomal origin display a great diversity. Two main characteristics can be drawn: the existence of several transcription units in a chromosomal organization unit and the possibility to point out a special architecture at the RNP fibril level. Applying a shadowing technique used for isolated molecules is an improvement compared with earlier methods based on PTA coloration.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.