Ribosome gradient analysis

Rabbit reticulocytes were incubated with isotopically labeled amino acids in the presence and absence of added hemin after incubation sucrose density gradient analysis was performed to determine the patterns of ribosomes and the radioactivity of the nascent, and completed polypeptide chains (Fig. 6) (Grayzel et al., 1966). The effects... 

Fig. 6. Sucrose density gradient analysis (Grayzel et al., 1966) of ribosomes from reticulocytes incubated with and without added hemin for 30 minutes and then with valine- C for an additional 2 minutes. Optical density, solid line radioactivity, dashed line.

G. A. Kowalchuk, J. R. Stephen, W. DeBoer, J. 1. Prosser, T. M. Embley, and J. W. Woldendorp, Analysis of ammonia-oxidizing bacteria of the beta subdivision of the class Proteobacteria in coastal sand dunes by denaturing gradient gel electrophoresis and sequencing of PCR-amplified I6S ribosomal DNA fragments. Appl. Environ. Microbiol. 6.1 1489 (1997). 

Fig. 10.21. Comparative analysis of nucleolar preribosomes by electrophoresis and sedimentation. Cells were labeled for 30 min with ( H)uridine (0.3 /jCi/ml, 1.2 /iM uridine, 4 x 10 cells/ml) and nucleoli prepared from which the preribosomes were extracted. A) one part of these preribosomes with some cytoplasmic ribosomes as markers were analysed by electrophoresis on a 2.2% uniform polyacrylamide gel B) Another part was analysed by sedimentation on a 5-20% sucrose gradient in EDTA-buffer...

Fig. 5.9. Slot-blot analysis of GAPDH mRNA from a sucrose gradient fractionation. Upper panel UV26o profiles of a cytoplasmic lysate in a 20-47% (w/w) sucrose gradient containing either 5mM Mgz+ (solid curve), or 10 mM EDTA (dotted curve). Lower panel Autoradiograph from the slot-blot analysis of fractions from both gradients hybridised with a probe specific for glyceraldehyde 3-phosphate dehydrogenase. Notice the altered sedimentation behaviour of GAPDH mRNA as a result of EDTA-mediated release from ribosomes.

Another approach to the analysis of mRNA transfer to the ribosomes is the study of cytoplasmic particles containing newly formed cytoplasmic mRNA. Leytin et al. (1971) have found that the material containing newly formed mRNA is somewhat less dense in the CsCl gradient than polysomes formed earlier. For example, while the bulk of polysomes have a buoyant density of 1.55 to 1.56 g/cm, the newly formed material possesses a density of 1.47 to 1.53 g/cm. Newly formed particles comprise only a small fraction of the total material since they cannot be detected by routine UV absorption methods. With more detailed analysis two newly formed components may be observed component A with p = 1.49 g/cm and component B with p = 1.52 g/cm. After long-term incubation, or in the presence of actinomycin D (chase experiment), almost all radioactive material is transferred from A to B peaks to the main peak of the polysomes. Inhibition of protein synthesis by cycloheximide interferes with these polysome transformations, and the label representing newly synthesized material accumulates in component A (p = 1.49 g/cm ). Thus one can suggest that component A converts to B and the latter is transformed into mature polysomes. It was found further than... 

Figure 6. Electrophoretic analysis of proteins associated with native 40 S ribosomal subunits of control and EMC-infected HeLa cells. Extracts were prepared from mock-infected and infected cells as described in Figure 2 0.5 ml of extract from control cells (b) or from cells infected 2 hr (a) or 3 hr (c) with EMC were layered on 15-50% sucrose gradients in 30 mM KC), 2mM Mg(OAc)p, 20 mM HHPES-KOH, pH 7 4i 1 mM dithiothreitol, and centrifuged 17 hr at 50f000 rpm. Fractions corresponding to the 40 S peak were combined, precipitated with 10% trichloroacetic acid, washed with acetone/ether (2/3) and ether, dissolved in sample buffer and fractionated on 12,5% polyacrylamide gels as previously described (14) The gels were stained with Goomassie Blue. Markers were run in parallel to assign M to the bands indicated by arrows.

Analysis of the cell-free translation system by sucrose gradient centrifugation, shows that PK-i causes a marked decrease in the amount of (55S-met-tRNA 4OS ribosome) complexes (12). Exogenous eIF-2 overcomes part of the dsRNA-dependent translational inhibition of mengo or globin mRNA in extracts from interferon treated cells (13) ... 

Proof of this assumption was provided by centrifuging in sucrose gradient a ribosome population prepared from reticulocytes. A number of peaks could be detected by ultraviolet spectrophotographic analysis at 260 mp. The preparations separated by gradient... 

Guan, L.L., Hagen, K.E., Tannock, G.W., et al. (2003) Detection and identification of Lactobacillus species in crops of broilers of different ages by using PCR-denaturing gradient gel electrophoresis and amplified ribosomal DNA restriction analysis. Appl Environ Microbiol 69, 6750-6757. 

Sucrose gradient profiles reveal that the preponderant component o each type of extract is ribosomal and that messenger RNA is small in amount. Since ribosomal RNA makes up the bulk of the mixed RNA, changes in the composition of the total following hormone treatment would be diflficult to detect by the usual methods of analysis. However, the nucleotide sequences of RNA synthesized by uterine... 

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