Affinity chromatography of Drosophila melanogaster ribosomal proteins to 5S rRNA.

The binding of Drosophila melanogaster ribosomal proteins to D. melanogaster 5S rRNA was studied using affinity chromatography of total ribosomal proteins (TP80) on 5S rRNA linked via adipic acid dihydrazide to Sepharose 4B. Ribosomal proteins which bound 5S rRNA at 0.3 M potassium chloride and were eluted at 1 M potassium chloride were identified as proteins 1, L4, 2/3, L14/L16, and S1, S2, S3, S4, S5, by two-dimensional polyacrylamide gel electrophoresis. Using poly A-Sepharose 4B columns as a model of non-specific binding, we found that a subset of TP80 proteins is also bound. This subset, while containing some of the proteins bound by 5S rRNA columns, was distinctly different from the latter subset, indicating that the binding to 5S rRNA was specific for that RNA species.

Isolation and characterization of cloned DNA sequences containing ribosomal protein genes of Drosophila melanogaster.

Ribosomal (r) proteins encoded by polyadenylated RNA were specifically precipitated in vitro from polysomes by using antibodies raised against characterized Drosophila melanogaster r proteins. The immuno-purified mRNA in the polysome complex was used to prepare cDNA with which to probe a D. melanogaster genomic library. Selected recombinant phages were used to hybrid select mRNAs, which were analyzed by in vitro translation. Three clones containing the genes for r proteins 7/8, S18, and L12 were positively identified by electrophoresis of the translation products in one-dimensional and two-dimensional polyacrylamide gels. Sequences encoding r proteins S18 and L12 were found to be present in the genome in single copies. In contrast, the polynucleotide containing the region encoding 7/8 may be repeated or may contain or be flanked by short repeated sequences. The sizes of mRNAs that hybridized to the recombinant clone containing 7/8 were significantly larger than would be expected from the molecular weight of protein 7/8, implying that there were unusually long 5' and 3' noncoding sequences. The mRNAs for r proteins S18 and L12 were however, only about 10% larger. In situ hybridizations to salivary gland polytene chromosomes, using the recombinant phage, revealed that the recombinant clone containing the gene for r protein 7/8 hybridized to 5D on the X chromosome; the recombinant clone containing the gene for S18 hybridized to 15B on the same chromosome, and the recombinant phage containing the gene for L12 hybridized to 62E on chromosome 3L. It is of interest that the genomic locations of all three r protein clones were within the chromosomal intervals known to contain the Minute mutations [M(1)0, M(1)30, and M(3)LS2]. Although each clone contained sequences specifying two to four proteins, none had more than one identifiable r protein gene, suggesting that different D. melanogaster r protein genes may not be closely linked.

Immunological evidence for structural homology between Drosophila melanogaster (S14), rabbit liver (S12), Saccharomyces cerevisiae (S25), Bacillus subtilis (S6), and Escherichia coli (S6) ribosomal proteins.

Specific antibodies directed against Drosophila melanogaster acidic ribosomal protein S14 were used in a comparative study of eucaryotic and procaryotic ribosomes by immunoblotting and enzyme-linked immunosorbent assays. Common antigenic determinants and, thus, structural homology were found between D. melanogaster, Saccharomyces cerevisiae (S25), rabbit liver (S12), Bacillus subtilis (S6), and Escherichia coli (S6) ribosomes.

Electron microscopic evidence for RNA polymerase loading at repeated sequences in non-transcribed spacers of D. virilis.

The organization of transcribed and non-transcribed sequences in transcriptionally active ribosomal DNA (rDNA) chromatin from Drosophila virilis nurse cells (excluding follicle cells) was examined by electron microscopy. Nuclear spread preparations revealed that the actively transcribing rRNA genes (rTUs) exhibited a single uniform length distribution (7 +/- 0.5 kb). Non-transcribed 'spacer' regions separating active rTUs were, on the other hand, quite heterogeneous in length with a mean of 1.95 +/- 1.4 kilobase pairs (kb). Within each 'spacer' segment of chromatin two morphologically distinct regions were discernable: (1) A fiber-free region adjacent to the 3' termination site of the preceding rTU which accounted for the majority of each non-transcribed 'spacer'. This region was highly heterogeneous in length, and was termed the 'Non-repetitive Spacer' (NRS). (2) A short region, largely uniform in length, which was located immediately 5' to each rTU. This region was covered by a linear array of RNA polymerase molecules with a few very short fibers. The arrangement of polymerase in these arrays and their proximity to rRNA coding sequences appeared to be morphologically indicative of multiple initiation sites which are thought to be present in this region of the non-transcribed spacer as evidenced by direct sequence analysis (Coen, E S & Dover, G A, Nucl acid res 10 (1982) 1017 and Miller, J R et al., Nucl acid res 11 (1983) 11) [6, 18]. The data suggest that the repeated sequences in the non-transcribed spacer region serve as multiple in vivo loading and/or initiation sites for RNA polymerase I. Our results further indicate that, although an estimated 75% of the ribosomal RNA genes in Drosophila virilis are interrupted by an intron in the 28Sb gene sequence (Barnett, T & Rae, P M M, Cell 16 (1979) 763) [1], none of those genes appear to be transcribed to any appreciable extent in the tissues examined. It is also reasonable to infer that the actively transcribing uninterrupted genes (and therefore the intron-containing genes as well) are clustered and not randomly interspersed.

Structural homology between Drosophila melanogaster and Escherichia coli acidic ribosomal proteins.

Antibodies raised against Drosophila melanogaster ribosomal proteins (r-proteins) were used to examine possible structural relationships between eukaryotic and prokaryotic r-proteins. The antisera were raised against either groups of r-proteins or individually purified r-proteins. Two antisera showed a cross-reaction with total Escherichia coli r-proteins in Ouchterlony double immunodiffusion assays: an antiserum against the D. melanogaster small subunit protein S14 (anti-S14) and an antiserum against a group of D. melanogaster r-proteins (anti-TP80). The specificity of the antisera and the identity of the homologous E. coli r-proteins were characterized by using immunooverlay and immunoblot assays. These assays indicated that anti-S14 was highly specific for protein S14 and anti-TP80 was a multispecific serum that recognized several of the D. melanogaster ribosomal proteins. The E. coli protein homologous to D. melanogaster protein S14 was identified as E. coli protein S6. By adsorption of the anti-TP80 serum, we determined that D. melanogaster protein 7/8 is homologous to the acidic E. coli protein L7/L12. D. melanogaster acidic protein 13 was also shown to be immunologically related to D. melanogaster protein 7/8.

Morphological analyses of active genes and chromatin.

Chromatin with nascent ribonuclear protein (RNP) fibers representing transcription of ribosomal DNA (top) and nonribosomal DNA (bottom). These two types of transcription can be distinguished on the basis of the size of transcription units, chromatin morphology and the inferred ratio of DNA packing, the frequency of RNP fibers (number of fibers per micrometer of chromatin), and the solitary v tandem repeat occurrence of fiber arrays. In some cases, the structure of RNP fibers is also distinctive for different transcription units. The micrographs presented herein, from Laird and Chooi, were taken of chromatin samples prepared similarly to the method described by Miller and Bakken. Cells and nuclei from Drosophila were ruptured in low ionic strength buffer; nuclear and cytoplasmic constituents were recovered on electron microscopic grids under conditions that facilitated spreading of chromatin and nascent ribonuclear protein fibers. Bar represents 1 micron.

Purification of Drosophila acidic ribosomal proteins.

The relatively acidic proteins (group A80) of Drosophila melanogaster ribosomes were separated by ion-exchange chromatography. Fractions containing one or more acidic proteins were combined into thirteen pools. The criterion for the combination was the migration pattern in one-dimensional polyacrylamide gels containing sodium dodecyl sulphate. Five proteins (7/8, S25/S27, S14, L1/L2 and L5/L6) required no further purification. The others were further purified as follows: proteins S7, and S9 by preparative gel electrophoresis: and protein 13 (to newly identified protein) by adsorption with conconavalin-A--agarose. Four proteins had no detectable contamination, and in each of the others the impurities were no greater than 3%. The amount of purified protein recovered from a starting amount of 2.63 g total 80-S ribosomal protein and a starting amount of 105 mg group A80 varied from 0.4 mg to 8.8 mg. The molecular weight of the proteins was estimated by polyacrylamide gel electrophoresis in sodium dodecyl sulphate. The amino acid composition of the individual purified proteins was determined. Several phosphorylated proteins were identified. Proteins 13b and 13c are phosphorylated derivatives of 13a; 7b/8b and 7c/8c are phosphorylated derivatives of 7a and/or 8a. Proteins 7/8 and 13 are distinct proteins but are very similar in amino acid composition.

Structure of ribosomes and ribosomal subunits of Drosophila.

The ultrastructure of Drosophila melanogaster cytoplasmic ribosomal subunits and monomers have been examined by electron microscopy. The Drosophila ribosomal structures are compared to those determined for other eucaryotes and E. coli. Negatively contrasted images of 60S subunits are seen in the most frequent view to be approximately round particles about 280 A in diameter. About 35% of the particles present a single prominent projection which we call the 60S peak. The peak emanates from a flattened region of the 60S subunit. The maximum observed length of the 60S peak is approximately 90 A. The Drosophila 60S peak is highly reminiscent of the E. coli L7/L12 stalk. The Drosophila 40S subunit is an elongated, slightly bent particle which measures 280 X 170 X 160 A. It bears a strong resemblance to small ribosomal subunits of other eucaryotes and is strikingly similar to the E. coli 30S subunit. Micrographs of 80S monomeric ribosomes show the long axis of the 40S to be parallel and in apparent contact with the flattened region of 60S subunit. The 60S peak appears to bisect the long axis of the 40S subunit. The 40S subunit seems to be oriented in the monomeric ribosome so that the 40S projection is toward the body of the large subunit. Comparison of our data with similar studies in different organisms indicates that the eucaryotic large ribosomal subunits exhibit morphological heterogeneity while the small subunits remain remarkably similar.

Decrease in ribosomal proteins 1, 2/3, L4, and L7 in Drosophila melanogaster in the absence of X rDNA.

The rRNA genes (rDNA) in Drosophila melanogaster are found in two clusters, one on the X and one on the Y chromosome. We have compared the ribosomal protein composition of wild-type Oregon-R flies containing both X-linked and Y-linked rDNA with that of flies containing only the Y-linked rDNA by two-dimensional polyacrylamide gel electrophoresis. Four basic proteins (1, 2/3, L4, and L7) normally present in wild-type flies were either electrophoretically not detectable (1, 2/3, and L4) or marginally detectable (L7) in flies with only Y-linked rDNA. No additional proteins were observed in these flies. However, immunodiffusion assays using specific antibodies raised against purified protein L4 confirmed that L4 was present but in relatively lower amounts in these Y-linked rDNA flies. An investigation was carried out to determine whether these electrophoretically undetectable proteins were more readily lost during ribosome preparation and hence were not readily detectable in the 80S particles by gel electrophoresis or whether they had been modified. Thus the proteins in the post-ribosomal cell supernatant and the high salt sucrose gradient were analyzed by two-dimensional gel electrophoresis and immunochemical assays with antibodies raised against protein L4 and total 80S ribosomal proteins. The experimental evidence indicates that there is a small amount of protein L4 and probably proteins 1, 2/3, and L7 in flies with only Y-linked rDNA but significantly less of these proteins than in wild-type flies.

Homology between Drosophila melanogaster and Escherichia coli ribosomal proteins.

Antibodies raised against D. melanogaster ribosomal proteins were used to examine possible structural relationships between eukaryotic and prokaryotic ribosomal proteins. The antisera were raised against either groups of ribosomal proteins or purified individual ribosomal proteins from D. melanogaster. The specificity of each antiserum was confirmed and the identity of the homologous E. coli ribosomal protein was determined by immunochemical methods. Immuno-overlay assays indicated that the antiserum against the D. melanogaster small subunit protein S14 (anti-S14) was highly specific for protein S14. In addition, anti-S14 showed a cross-reaction with total E. coli ribosomal proteins in Ouchterlony double immunodiffusion assays and with only E. coli protein S6 in immuno-overlay assays. From these and other experiments with adsorption of anti-S14 with individual purified proteins, the E. coli protein homologous to the D. melanogaster protein S14 was established as protein S6.

An electron microscopic method for localization of ribosomal proteins during transcription of ribosomal DNA: a method for studying protein assembly.

We describe a method for the localization of ribosomal proteins on electron microscopic spreads of active rRNA genes. The method consists of raising antibodies against Drosophila melanogaster proteins and allowing these antibodies to react with lysates of D. melanogaster egg chambers. The locations of the bound IgGs in the active transcripts are detected with goat anti-rabbit IgGs that have been labeled with electron-dense polymethacrylate spheres. By statistical analysis we can generate a confidence interval for the initial point of protein assembly for a particular protein. The first point of protein assembly for S14 is located near the 5' end of the pre-18S rRNA. In contrast, the first point of protein assembly for L4 is at 0.38 unit from the initiation point (a unit being the length of a ribosomal transcription unit). The binding patterns of S14 and L4 are consistent with the 5' proximal and the 5' distal orientations of the pre-18S and the pre-28S rRNAs. The method described here provides an approach to the elucidation of the assembly of eukaryotic ribosomal proteins in vivo.

A comparison of the ribosomal proteins of Drosophila ovary, adult, and embryo.

The proteins in the 80S ribosomes of Drosophila melanogaster ovaries and adults have been characterized by two-dimensional polyacrylamide gel electrophoresis. When ribosomal proteins of ovaries and adults were compared with those from embryos, all 3 tissues showed a similar number of proteins. In addition, qualitatively, the electrophoretograms of proteins extracted from the ribosomes of these 3 tissues were found to be indistinguishable. However, apparent quantitative differences in certain acidic proteins were observed between tissues. Using ribosomes from embryos as a standard for comparison, ribosomes from adult flies that were more than 14 days old appeared to have relatively larger amounts of acidic proteins S7 and S9, and relatively smaller amounts of acidic proteins S14 and S25/S27. The transition period occurred during the ninth to thirteenth day of adult fly development. Significant differences were not detected between ovarian and embryonic acidic ribosomal proteins. In contrast to the differential ratio of acidic proteins in ovaries, adults, and embryos, a similar distribution of basic proteins was found in these tissues.

The in vivo expression of pseudo ribosomal RNA genes in Drosophila melanogaster.

Electron microscopic examination of chromatin has indicated that there are two major classes of ribosomal transcription units (rTUs) in Drosophila melanogaster. (a) a standard class which is about 8 kb in length and (b) a longer class (up to 15 kb) which has been hypothesized as having an intron in the 28S region of the gene. On rare occasions, we have observed a third class of TUs, which we term pseudo rTUs (or pseudo ribosomal RNA genes), distributed among the standard rTUs. Pseudo rTUs are composed of short fiber arrays with clear gradients in their RNP fiber lengths. The pseudo rTUs observed in egg chambers and blastoderm embryos are 4.1 +/- 0.58 kb in length and are found in tandem with non-transcribed spacer regions. The lengths of the non-transcribed spacer regions are found in 2 classes: one at 4.94 +/- 1.78 kb and the other at 19.28 +/- 2.11 kb. The present information suggests that these pseudo rTU fiber arrays are ribosomal in origin as their RNP fibers cross-react with antibodies raised against Drosophila ribosomal proteins. The pattern of distribution of D. melanogaster ribosomal protein L4 on RNP fibers in standard rTUs is compared with that in pseudo rTUs. This was determined by a novel approach involving the use of electron microscopic spreads of rTUs to which had been added IgGs labelled with polymethacrylate spheres. Pseudo ribosomal RNA genes are present in both the X (6%) and in the Y (6%) ribosomal chromatin as is indicated by their existence in nurse cells of both Oregon-R females, and females of the genotype sc4sc8/sc4sc8/y+Y.

Purification of Drosophila ribosomal proteins. Isolation of proteins S8, S13, S14, S16, S19, S20/L24, S22/L26, S24, S25/S27, S26, S29, L4, L10/L11, L12, L13, L16, L18, L19, L27, 1, 7/8, 9, and 11.

The proteins of Drosophila melanogaster embryonic ribosomes were separated into seven groups (A80 through G80) by stepwise elution from carboxymethylcellulose with lithium chloride at pH 6.5 by procedures previously described [Chooi, W. Y., Sabatini, L. M., MacKlin, M. D., & Fraser, W. (1980) Biochemistry 19, 1425-1433]. Three relatively acidic proteins, S14, S25/S27, and 7/8, have now been isolated from group A80 by ion-exchange chromatog raphy on carboxymethylcellulose eluted with a linear gradient of lithium chloride at pH 4.2. Fractions containing the relatively basic proteins (groups B80 through G80) were furher combined into a total of 24 "pools". The criterion for combination was the migration patterns in one-dimensional polyacrylamide gels containing sodium dodecyl sulfate (NaDodS04) of every fifth fraction from the carboxymethylcellulose column. Each pool contained between 1 and 12 major proteins. Proteins S8, S13, S16, S19, S20/L24, S22/L26, S24, S26, S29, L4, L10/L11, L12, L13, L16, L18, L19, L27, 1, 9, and 11 have now been isolated from selected pools by gel filtration through Sephadix G-100. The amount of each protein recovered from a starting amount of 1.8 g of total 80S proteins varied form 0.2 to 10.8 mg. Five proteins had no detectable contamination, and in each of the others the impurities were no greater than 9%. The amino acid composition of the individual purified proteins was determined. The molecular weights of the proteins were estimated by polyacrylamide gel electrophoresis in NaDodSO4.

Group fractionation and determination of the number of ribosomal subunit proteins from Drosophila melanogaster embryos.

Proteins were extracted from ribosomes and (for the first time) from ribosomal subunits of Drosophila melanogaster embryos. The ribosomal proteins were analyzed by two-dimensional polyacrylamide gel electrophoresis. The electrophoretograms displayed 78 spots for the 80S monomers, 35 spots for the 60S subunits, and 31 spots for the 40S subunits. On the basis of present information, we propose what we believe to be a reliable and convenient nomenclature for the proteins of the ribosomes and each of the subunits. A pair of acidic proteins from D. melanogaster appears to be very similar in electrophoretic mobility to the acidic proteins L7/L12 from Escherichia coli and L40/L41 from rat liver. The electrophoretogram of proteins from embryonic ribosomes shows both qualitative and quantitative differences from those of larvae, pupae, and adults previously reported by others. The proteins of the 40S subunit range in molecular weight from approximately 10,000 to 50,000, and those from the 60S subunit range from approximately 11,000 to 50,000.

The occurrence of long transcription units among the X and Y ribosomal genes of Drosophila melanogaster: transcription of insertion sequences.

Most of the ribosomal transcription units (rTUs) in Drosophila melanogaster observed by electron microscopy measure about 8 kb; a length which corresponds to the size of the 38S precursor to ribosomal RNA in D. melanogaster. However, interspersed among these rTUs are transcription units that are much longer (up to 14.6 kb) than the 8 kb expected for rTUs. Some of these larger length estimates can be attributed to stretching but an important fraction is significantly larger and has up to 60 more fibers per gene.--The following evidence suggests that these larger transcription units are ribosomal genes consisting of insertion sequences. The long transcription units are within the sizes expected for rTUs containing insertion sequences as reported by other workers. Their RNP fibers cross-react with antibodies raised against ribosomal proteins in a manner similar to that observed for ribosomal RNP. They are interspersed among rTUs in the X chromosome.--These putative ribosomal genes carrying insertions are present both in the X and, although to a lesser extent, in the Y ribosomal chromatin as is indicated by their existence in nurse cells of both Oregon R females and females of the genotype sc4sc8/sc4sc8/y+ Y. Analysis of the fiber patterns of "long TUs" supports the hypothesis that the insertion region is being transcribed.--"Long TUs" are found in tandem with non-transcribed spacer regions which are heterogeneous in length with a mean of 1.53+/-0.61 micrometers (or 8.5+/-3.4 kb).

Analysis of chromatin-associated fiber arrays.

Electron microscopic examination of chromatin from embryonic nuclei of Oncopeltus fasciatus and Drosophila melanogaster reveals arrays of chromatin associated fibers. The lengths and spacings of these fibers were analyzed to provide a basis for defining and interpreting regions of transcriptionally active chromatin. The results of the analysis are consistent with the interpretation of some fibers as nascent RNA with associated protein (RNP). The chromatin segments underlying these fiber arrays were classified as ribosomal or non-ribosomal transcription units according to definitions and criteria described by Foe et al. (1976). Nascent fibers on active ribosomal transcription units were analyzed and compared for Drosophila melanogaster, Triturus viridescens, and Oncopeltus fasciatus. A common feature of the fiber patterns on ribosomal TUs is that origin-distal fibers exhibit greater length variability and a lower slope relative to proximal fibers. The region of increased variability in fiber lengths is correlated with the expected location of 28S ribosomal RNA sequences in the distal half of each ribosomal transcription unit. Because 28S ribosomal RNA appears to contain more extensive regions of base sequence complementarity, we suggest that the length of ribosomal RNP fibers is influenced under our spreading conditions by the secondary structure of the nascent RNA. In order to calculate the RNA content of RNP fibers, chromatin morphology was used to estimate lengths of transcribed DNA. The packing ratio of DNA in chromatin, which we express as the length of B-structure DNA divided by length of chromatin, is 1.1-1.2 and 1.6 for the DNA in active ribosomal and non-ribosomal chromatins, respectively. These DNA packing ratios are used to determine the extent to which nascent RNP fibers are shorter than the transcribed DNA (expressed as DNA/RNP length ratio). For non-ribosomal transcription units and for proximal fibers of ribosomal transcription units. DNA/RNP length ratios are relatively constant within each array. However, considerable variability in this ratio (4-23) is observed for different arrays of fibers. Possible sources of this variability are considered by comparing ratios derived from the presumably identical ribosomal transcription units. Further analysis of the morphology of nascent fibers may elucidate the contributions of proteins and successive RNA sequences to RNP structure.

Morphology of transcription units in Drosophila melanogaster.

We have used an electron microscopic analysis to define and to characterize active transcription units of Drosophila melanogaster. The lengths and spacings of nascent ribonuclear protein (RNP) fibers were determined on embryonic chromatin that was spread using techniques introduced by Miller and Beatty (1969). The data are consistent with the occurrence of specific sites of transcription initiation and termination. We apply the term transcription unit (TU) to a chromatin region bounded by these control sites. Two classes of TUs are active in Drosophila melanogaster embryonic cells--those synthesizing ribosomal RNA and those synthesizing non-ribosomal RNA. The classes can usually be distinguished on the basis of TU size, chromatin morphology and inferred DNA packing ratio, frequency of RNP fibers (number of fibers per mum of chromatin), and the solitary vs. tandem repeat occurrence of fiber arrays. The results indicate that non-ribosomal transcription units have lengths in accord with the expectation that DNA of each chromomere is transcribed as a unit. Some nascent fiber arrays in D. melanogaster have more complex patterns of RNP fiber lengths. We suggest that these are a consequence of cleavage of RNP fibers at specific sites during transcription. These sites of transcriptional control and the amounts of DNA between them provide a basis for further relating units of transcription to units of gene function.