We have undertaken an immunological and biochemical analysis of the most abundant soluble protein of previtellogenic Xenopus oocytes, 42S p48. We show that this protein shares immunological cross‐reactivity with elongation factor 1 alpha (EF‐1 alpha). Direct assays of both 42S fractions and purified 42S p48 show that this cross‐reactivity is of functional significance since 42S p48, like EF‐1 alpha, can transfer charged amino acids to ribosomes. We further demonstrate that 42S p48 is degraded soon after the onset of vitellogenesis, while the EF‐1 alpha concentration remains essentially unchanged during this transition. These properties of 42S p48 are discussed with regard to its role in oogenesis.
Mouse polyclonal antibodies have been raised against two human proteins (IEF [isoelectric focusing] 31, Mr = 50,000; IEF 46, Mr = 43,500) that have previously been shown to be present in HeLa cytoskeletons enriched in intermediate-sized filaments. Immunoprecipitation studies show that both proteins share common antigenic determinants with each other and with the putative human keratins IEF 36 and 44, also present in HeLa cytoskeletons. Indirect immunofluorescence studies showed that both antibodies revealed similar filamentous networks in various cultured epithelial cells of human origin. These included AMA (transformed amnion), HeLa (cervical carcinoma), normal amnion cells, FI-amnion (transformed amnion), WiSH-amnion (transformed amnion), Chang liver (liver), and Detroid-98 (sternal marrow). Human cells that did not react with both antibodies included skin fibroblasts, lung fibroblasts (Wl-38), SV40-transformed lung fibroblasts, Molt 4 (leukemia), lymphocytes, and monocytes. These results were in complete agreement with the presence or absence of both proteins in two-dimensional gels of the different cell types. Exposure of AMA cells to demecolcine (24 h; 10 gg/ml) caused the total collapse of vimentin filaments but, as seen by indirect immunofluorescence, caused only a partial redistribution of the IEF 31 and 46 filaments. These results are taken to suggest that both proteins are components of the intermediate-sized filaments of the "keratin" type. The antibodies could be clearly differentiated by staining human bladder carcinoma EJ 19 cells, as only the IEF 46 antibody stained a filamentous network in these cells. The occurrence of keratins IEF 31, 36, 44, and 46 in different cultured human epithelial cells has been studied using two-dimensional gel electrophoresis.Epithelial cells including keratinocytes contain intermediate filaments (7-1 l-nm thick fdaments) that are related to keratins of the epidermis as judged by cross-reactivity using various keratin antibodies (11, 13-17, 20, 23-26). Franke et al. (16) have proposed the term "cytokeratins" to describe the proteins that compose these fdaments and have presented evidence for a diversity of these proteins in various epithelial cells and tissues (12). Recently, we showed that cytoplast skeletons from epithelial HeLa cells enriched in intermediate filaments exhibited, besides virnentin, a few proteins that were likely candidates for keratins. In this study we show that mouse poly-416 clonal antibodies raised against human proteins IEF (isoelectric focusing) 31 (Mr = 50,000) and IEF 46 (Mr = 43,500 (HeLa catalogue numbering system; 3, 5) react with intermediate filaments of the "keratin" type in a variety of human cultured cells of epithelial origin. Using a sensitive immunoprecipitation assay, we further show that both keratins share common antigenic determinants with each other and with the putative human keratins IEF 36 and 44 (3,5,7,8,16). The occurrence of the four keratinlike proteins in different cultured human epithelial cells has been d...
Two-dimensional gel electrophoretic (NEPHGE) analysis of proteins from mouse 3T3B and 3T3B/SV40 cells labelled with [methyk3H]methionine in the presence of cycloheximide have revealed that the elongation factor la (EF-lcu) in these cells is methylated and that the extent of methylation is higher in the SV40 transformed cell type. It is suggested that methylation may account for differences in growth properties for the different cell types.Elongation factor Iru Two-dimensional gel electrophoresis Methytation 3 T3B 3T3B/SV40
Abstract. We have isolated the cDNA for 42Sp48 and EF-la from mixed stage oocytes and tailbud (stage 22) Xenopus laevis cDNA libraries by use of the cDNA for human elongation factor-ltx (EF-lo0 as probe. The nucleotide and deduced amino acid sequences of the entire coding region of 42Sp48 and EF-hx cDNA were established. The proposed functional homology of the proteins is reflected in highly conserved amino acid sequences (91% identity), while the large number of silent mutations at the gene level may serve to prevent recombination at their loci. 42Sp48 is apparently encoded by two genes in Xenopus, while no sequences corresponding to 42Sp48 could be found in murine or human genomic DNA. 42Sp48 has been proposed to act as a stage-specific elongation factor in Xenopus. Comparison of the deduced amino acid sequences of 42Sp48 and EF-lo~ with that of elongation factor Tu from E. coli, for which the three-dimensional structure including that of the GTP binding site have been determined, supports this hypothesis.OGENESIS in amphibians can be divided into two distinctive stages; previteLlogenesis, in which the cytoplasm of the cell is devoid of yolk and is transparent, and viteUogenesis, in which yolk, lipids, glycogen, and ribosomes rapidly accumulate. An oocyte of the African clawed toad, Xenopus laevis, contains extremely large amounts of RNA, 95 % of which is ribosomal in origin and 2 % of which is tRNA, while the remainder is thought to be mRNA (Brown and Littna, 1964). The oocyte transcribes 5S rRNA and tRNA genes constantly during all stages of growth and during previtellogenesis, in which the amplified 28S and 18S rRNA genes are not transcribed, 80% of the total RNA content comprises 5S and tRNA species (Ford, 1971). These components are stored in two cytoplasmic ribonucleoprotein particles, the 7S and 42S ribonucleoprotein particles (Denis
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