Showing papers in "Molecular Biology Reports in 1986"

UV crosslinking of RNA to nylon membrane enhances hybridization signals.

Abstract: An improvement in the detection by nucleic acid hybridization of size-fractionated RNA immobilized to nylon-based membranes is described. Electrophoretic transfer of RNA to nylon membranes permits a quantitative determination of different RNA transcripts on the same membrane after sequential hybridization using different 32P-labeled DNA probes. UV corsslinking of the RNA to the nylon membrane increased the intensity of the radioactive signals. Using the method reported here, increased signals of between 10 and 40 fold were observed, depending on the species of transcript tested. Moderately abundant as well as rare transcripts can easily be detected in as little as 5 μg total cellular RNA.

Characterization of human c-fes/fps reveals a new transcription unit (fur) in the immediately upstream region of the proto-oncogene.

Abstract: Comparison of nucleotide sequence data of the 5' region of a fes/fps viral oncogene with those of the v-fes/fps homologous regions of man and cat revealed the position of the 3' portion of an as yet unidentified c-fes/fps exon. Comparative Southern blot and heteroduplex analysis of human and feline DNA immediately upstream of the v-fes/fps homologous regions showed extensive but discontinuous homology over a 9 kbp DNA stretch, which we have designated as fur. Northern blot analysis of mRNA from KG-1 myeloid cells with fes/fps- or fur-specific probes revealed a 3.0 kb fes/fps and a 4.5 kb fur transcript. Analysis of a number of tissues of an adult Wistar Lewis rat for the presence of fur transcripts revealed its differential expression pattern. An 0.95 kbp fes/fps-related and a 2.2 kbp fur-related cDNA recombinant clone were isolated from an oligo(dT)-primed KG-1 cDNA library. Comparative nucleotide sequence analysis of the fes/fps cDNA and its human genomic counterpart indicated that the cDNA contained genetic sequences that were identical to and colinear with exon 15-19 and, furthermore, that the poly(A) addition signal near the 3' end of exon 19 was functional. Similar analysis of the 2.2 kbp fur cDNA indicated that the poly(A) addition signal of the fur transcript was in close proximity of the newly discovered fes/fps exon. The region in between contained a CATT sequence but no 'TATA' box. The fur transcript was characterized by a long noncoding region at its 3' end.

Synthesis of somatomedin C/insulin-like growth factor I by human placenta.

Abstract: We have reported the presence of insulin-related poly A+RNA sequences in human placenta by RNA to DNA hybridization. In this study we have used a monoclonal antibody to somatomedin C/insulin-like growth factor I (Sm-C/IGF-I) to identify somatomedin-like proteins whose synthesis is directed by placental mRNA. Poly A+RNA from first trimester and term placenta was translated in a cell-free system using micrococcal nuclease-treated reticulocyte-lysate and [35S]methionine as a label. From 2.0×106 cpm of specifically incorporated [35S]methionine labeled protein, an immunoprecipitate with an apparent molecular weight of 14000 represented about 0.1% of total radioactivity in the translational products of poly A+RNA of first trimester placenta. A less prominent band (0.006%) of the same apparent molecular weight was also evident from translational products of term placental mRNAs. This protein could be competed with either acromegalic serum or synthetic Sm-C/IGF-I when added prior to immunoprecipitation. Translational products synthesized from mRNA of term placenta showed a second labeled band of 24000 daltons. This band was less effectively competed by acromegalic serum and not competed with either Sm-C/IGF-I or IGF-II and therefore its identity is uncertain. A protein similar to Sm-C/IGF-I is, therefore synthesized in first trimester placenta and to a lesser extent at term, suggesting developmental changes in Sm-C/IGF-I synthesis. Because Sm-C/IGF-I may act in a paracrine fashion, our findings suggest a role for Sm-C/IGF-I in growth of the placenta during early gestation.

Differential polyadenylation pattern of ovalbumin precursor RNAs during development.

Abstract: The expression of the ovalbumin gene encoding for the major hen oviduct protein slows down with age. Analysis of Northern blots of electrophoretically separated total and poly(A)+ RNA from oviducts of hens of different age with an ovalbumin-specific probe (nick-translated 9.5 kb ovalbumin gene DNA cloned into pBR322) revealed that the largest high molecular weight ovalbumin RNA precursor (7.9 kb band, representing the putative primary transcript of the ovalbumin gene) was most intense if total RNA from non-egg-laying old hen oviduct was checked as compared to that from egg-laying mature animals. On the other side, the 7.9 kb RNA precursor band was readily detected in the poly(A)+ RNA from mature hen oviduct where-as it was invisible in the old hen oviduct poly(A)+ RNA fraction. The lack of detection of the 7.9 kb RNA species within the poly(A)+ RNA fraction and its increased concentation within the total oviduct RNA, both from old animals, suggest that age-dependent impairment of ovalbumin mRNA processing may be caused by altered polyadenylation of distinct RNA precursors.

Direct in vitro action of thyroid hormones on mitochondrial RNA-polymerase

Abstract: The authors show the direct in vitro action of thyroid hormones on RNA-polymerase activity in rat liver mitochondria. 3,5,3′ L-triiodothyronine (L-T3) and 3,5,3′,5′ L-tetraiodothyronine (L-T4) stimulate mitochondrial RNA synthesis without either increasing the permeability of preswollen mitochondria or stimulating the synthesis of the triphosphate ribonucleotides (NTP's). Thyroid hormones do not directly depress mitochondrial RNA hydrolysis. Studies carried out with structural analogues of thyroid hormones indicate the structural specifications of the regulating system of the mitochondrial RNA-polymerase. L-T3 and L-T4 are also effective ‘in vitro’ on mitochondria obtained from animals undergoing different hormonal and dietary treatments, with the exceptions of those fed with a hypoprotein diet. Thus, the authors suggest the possible intervention of a specific mitochondrial receptor for L-T3 and L-T4.

The localization of a lectin-like component on the Leishmania cell surface.

Abstract: The binding between marcrophage-like cells J774G8 and Leishmania braziliensis (NR) promastigotes was studied ‘in vitro’ by a radioisotopic assay under various conditions in the absence of serum Different sugars, N-acetyl-D-glucosamine, D-glucose, D-mannose, D-galactose, and chitin, diminished the binding of the parasite, whereas other sugars, D-arabinose, D-fucose and D-xylose, did not affect the binding The presence of a lectin-like ligand specific for N-acetyl-D-glucosamine has been detected on the cell surface of the Leishmania braziliensis (NR) by fluorescence microscopy

Cloning and structure of a mouse interleukin-2 chromosomal gene

Abstract: Using non-stringent hybridization with a human interleukin-2 cDNA probe, we have isolated recombinant phages from a mouse genomic DNA library cloned in the EMBL3 phage The sequence and organization of the mouse interleukin-2 (IL-2) gene was determined By comparison with the human IL-2 sequence, three introns can be identified with lengths of 99, ±2 400, and ±1 900 base pairs, respectively The mouse IL-2 gene codes for a polypeptide of 169 amino acids and contains a putative signal peptide of 20 amino acids The homology to the human interleukin-2 is 72% at the nucleotide level in the coding part and 65% at the amino acid level An extraordinary sequence, consisting of 12 consective CAG codons coding for glutamine, is found in the first exon

A study of mitochondrial ribosomes from the higher plant Solanum tuberosum L.

Abstract: Ribosomal subunits are isolated from potato tuber mitochondria devoid of contaminating organelles. The sedimentation constants of the two mitochondrial ribosomal subunits are 33S and 50S respectively. The apparent sizes of the high molecular weight RNAs are 19S and 25S. The proteins of these ribosomes have been analyzed by two-dimensional electrophoresis in SDS polyacrylamide gels to determine their number and molecular weights. The small subunit contains 35 protein species ranging from 8 to 60 kDa. The 50S large subunit contains 33 protein species ranging from 12 to 46 kDa. These preliminary results are the first analysis made on mitochondrial ribosomes from a higher plant.

Effects of DNA methylation on specific transcription by RNA polymerase II in vitro.

Abstract: In vitro transcription in a HeLa cell lysate by RNA polymerase II directed by a chicken feather keratin gene promotor has been studied using unmethylated template DNA and DNA methylated in vitro by HpaII methylase. The efficiency of specific gene transcription from methylated DNA was dependent on topology of the input DNA, the most significant effect being complete inhibition of transcription from one template which contained three methylation sites, one just 5′ and two greater than 500 bases 3′ to the site of transcription initiation. The inhibition of transcription depends on a factor(s) which is variably present in lysate preparations and is labile on storage at -70°.

In vitro release of α1-acid glycoprotein RNA sequences shows fidelity with the acute phase response in vivo

Abstract: The acute phase reaction of rat liver to subcutaneous turpentine challenge results in a 20- to 100-fold increase in α1-acid glycoprotein (αAGP) mRNA. We utilized this response to establish conditions appropriate for study of RNA transport in vitro using hybridization with 32P-labeled exon and intron αAGP sequences. Contamination of nuclear preparations by membrane-absorbed cytoplasmic RNA was eliminated by detergent-rinsing. The in vitro incubation conditions that most reflected the in vivo state required RNase inhibitor (purified from placenta), polyvinylpyrrolidone to prevent nuclear swelling, and addition of ATP. Under these circumstances, αAGP sequences were transported only from turpentine-stimulated preparations, were found only in poly(A) + RNA, and were the same size as authentic cytoplasmic mRNA. Omission of polyvinylpyrrolidone resulted in release of some αAGP sequences in smaller, more heterogeneous poly(A)-RNA, and leakage of some αAGP sequences was observed from control preparations. Omission of ATP resulted in restriction of mature αAGP mRNA to the nucleus. In contrast to αAGP mRNA, transport of albumin mRNA was decreased 3-4X in turpentine-treated preparations. The largest αAGP intron was not found in RNA transported from treated nuclei in complete medium. The intron-containing fragments remained in the nucleus, largely in poly(A)- RNA of a size consistent with free intron. Some hybridization of intron sequences was observed with cytoplasmic and nuclear membrane-associated poly(A) + RNA preparations which may represent 3′-processing catabolites; leakage of these sequences was considerably greater in the absence of PVP. On the basis of densitometric estimates, a 5-fold increase in the amount of αAGP exon sequences was observed in nuclear RNA, comparing treated with control animals, but transport of αAGP exon sequences was detectable only from treated nuclei, indicating at least a 50-fold increase in abundance of αAGP sequences. This suggests that a selective gating mechanism may be operative at the level of post-transcriptional nucleocytoplasmic transport during induction of αAGP in the acute phase response.

Studies on the local activity of antiandrogens at the molecular and histological level.

Abstract: Androgen dependent skin disorders are important in clinical practice. Effective topical antiandrogens would lead to a breakthrough in their treatment. Although many attempts have been performed to develop such compounds, major successes have not been forthcoming. In the present study three existing antiandrogenic molecules have been compared with regard to their effect on androgen metabolism, receptor competition and on histological parameters in the hamster flank organ test. It appears that the effect on the hamster pigmented spot can be predicted on the basis of molecular mechanism. However, the effects on histological parameters are apparently dependent on additional factors such as metabolism of the active substance before reaching the sebaceous structure or limited penetration through the skin surface. The results indicate that in the development of new antiandrogens pre-screening can be performed with the aid of metabolic and receptor studies, while the histological parameters in the hamster flank organ test provide an animal model with a good predictive value.

Identification of the substrates of the casein kinase II associated with non-polysomal messenger ribonucleoproteins of A. salina cryptobiotic embryos.

Abstract: The association of a protein kinase with cytoplasmic non-polysomal messenger ribonucleoproteins is demonstrated by chromatography on oligo(dT)-cellulose and sucrose gradient centrifugation The cAMP-independent enzyme is inhibited by caffeine and poly(L)-glutamic acid and is classified as a casein kinase II Among the exogenous proteins initiation factor eIF2 is the best substrate and is 78 times more efficiently phosphorylated than casein Endogenous mRNP protein substrates have a Mr of 125000, 65000, 38000, 26000 and 23500 The main phosphate acceptor is the Mr38000 poly(A)-binding protein Dephosphorylation of the poly(A)-binding protein by protein phosphatases decreases its RNA binding property The effect of phosphorylation-dephosphorylation of mRNP proteins on the initiation of protein synthesis is discussed

Human hair follicle cells in culture: the development of a new culture system and its potential applications

Abstract: Of all forms of cancer in man 80 percent are carcinomas, i.e. tumors which arise from epithelial tissue. The latter consists of more or less polygonal cells and forms the lining of skin, intestine and trachea. For this reason, research upon the origin of many forms of cancer should be performed on epithelial cells (2, 12, 45). One of the experimental approaches is exposure of those cells to carcinogenic substances. However, for ethical reasons this cannot be done on test persons, but must be performed in tissueor in cell culture. One has to keep in mind that human and animal cells have different sensibilities for carcinogens, resulting in different risks when exposed to the same carcinogens (38). The use of human cells is restricted: for practical reasons mostly cells of blood and skin will be explored. Blood cells, however, are not of epithelial origin. Thus keratinocytes, the epithelial cells of skin, should be preferentially utilized.

Evidence that the methylation inhibitor cycloleucine causes accumulation of a discrete ribosomal RNA precursor in hamster mitochondria.

Abstract: A novel RNA fraction, 'Cy RNA,' that accumulates in mitochondria when hamster cells are treated with the methylation inhibitor cycloleucine, has been characterized by high resolution acrylamide gel electrophoresis and DNA-RNA hybridization. Cy RNA ran in gels as a discrete band, with an apparent chain length of 2 600. It hybridized specifically to restriction fragments containing genes for the mitochondrial ribosomal RNAs. We infer that Cy RNA is a discrete polycistronic ribosomal RNA precursor transcript, whose processing is dependent on normal methylation.

Viscosity of chromatin solutions increases with increasing ionic strength.

Abstract: Increasing the ionic strength of rat liver chromatin solutions above 0.4 M causes increasing viscosity. This indicates transformation of the compact chromatin molecules to more elongated forms. In the range of 0.4-0.5 M ionic strength histone H1 is dissociating continuously from the chromatin and the quaternary structure chromatin unravels. At ionic strength higher than 0.5 M the viscosities of chromatin solutions are furthermore increasing due to structural deformation. Near 0.7 M ionic strength the core histones H2A and H2B begin to dissociate from the chromatin, and the opening of the nucleosome cores leads to increasing elongation of the chromatin molecules.

Sequence analysis of the larval beta II-globin gene of Xenopus laevis.

Abstract: The 1822 bp sequence of the larval Xenopus laevis βII-globin gene is reported together with 240 bp upstream of the gene and 190 bp beyond the site of polyadenylation. The mRNA start point was determined by primer extension as well as nuclease S1 mapping and the polyadenylation site by comparison of the gene sequence to the mRNA sequence derived from a corresponding cDNA clone. Like other vertebrate globin genes, this gene comprises three exons interrupted by two intervening sequences (IVS). IVS I spans over 582 nucleotides and interrupts the exon sequences within codon 30. IVS II is located between the codons 104/105 and spans over 617 nucleotides. The 5′ region of the gene contains the canonical TATAA homology at position-31. Comparison of the upstream sequence to that of Xenopus laevis larval βII-globin gene revealed a conserved sequence, located between nucleotide positions-60 and-87, which might function as regulatory element of transcription. Whereas the upstream region of the larval βII-globin gene does not contain a CAAT box, we notice a reiterated AAATGA motif and discuss its possible significance.

The role of interfacial structured water on the glycoprotein arrangement in liposomes.

Abstract: The effect of perturbing the interfacial water structure in liposomes on the glycoprotein arrangement in the bilayer was investigated. This perturbation was achieved by a series of reagents called structure makers and breakers. The glycoprotein arrangement in the liposomes was determined by fluorescence measurement with 1-anilino-2-naphtalene sulphonate (ANS). A dependence of (n) (number of binding sites for ANS on the glycoprotein molecule) with concentration of structure maker and breaker reagents was observed. The results have been interpreted as a possible new arrangement of membrane-bound glycoprotein, due to the effect of perturbing the interfacial water structure in the liposomes.

Osmotic water permeability through liposomes in the presence of α1-acid glycoprotein

Abstract: α1-acid glycoprotein (orosomucoid) from human blood serum was isolated in pure form and then reconstituted into large multilamellar liposomes, consisting of a binary mixture of hen-egg phosphatidylcholine and cholesterol. These liposomes were found to be osmotically sensitive. The osmotic water permeability of proteoliposomes was determined by light-scattering measurements of the osmotic volume changes after mixing with hyperosmotic solutions of potassium salts and aminoglycoside antibiotics. The initial rate of water outflow was measured as a function of glycoprotein concentration in the mixture for the preparation of proteoliposomes. This can serve as an indication for membrane permeability to the solutes used in these experiments. It was shown that aminoglycoside antibiotics passed much faster across the membrane than potassium salts, in the presence of glycoprotein in the liposomes. A recognition pattern in the osmotic behavior of these proteoliposomes was assumed.

Correlations of repetitive and AT-rich DNA segments within the chicken globin gene domains

Abstract: The repetitive DNA segments were mapped within a 30 Kbp genomic domain including (in 5′ to 3′ order) the chicken embryonic pi and adult alpha D (minor) and alpha A (major) globin genes. Two repeats map 5 and 8 Kbp upstream from the embryonic pi gene and another 3 Kbp downstream of the adult alpha A gene. These repetitive DNA sequences are placed within, or immediately adjacent to the AT-rich DNA segments framing this domain. Similar correlations exist also within the chicken beta globin gene domain. The positions of these AT-rich and repetitive DNA segments framing the alpha globin gene domain also correlate with other already explored features of long range DNA organisation, as clusters of sites of DNAse I hypersensitivity and differential methylation, sites of Matrix-DNA attachment, and with the beginning and end of the transcribed domain.

Changes in the pattern of histone H1 phosphorylation during Drosophila hydei embryogenesis.

Abstract: Histone H1 phosphorylation was examined during embryonic development of Drosophila hydei. A changing pattern of H1 phosphorylation upon separation on an acid-urea polyacrylamide gel was observed in the course of Drosophila embryogenesis. It is considered to be related to the decrease of the mitotic activity of the cells as development proceeds.

Study of chromatin structure in ataxia-telangiectasia cells

Abstract: Micrococcal nuclease was used as a probe to study chromatin structure in control and ataxia-telangiectasia cells. The rate and extent of release of acid-soluble nucleotide was similar in both cell types. Production of mono- and oligonucleosomes by micrococcal nuclease as determined by gel electrophoresis also failed to reveal differences in chromatin structure between control and ataxia-telangiectasia cells. Radiation exposure did not significantly alter the kinetics of digestion. These results indicate that there are no gross alterations in chromatin structure in ataxia-telangiectasia cells.

Psoriatic hair follicle cells. IV. Calmodulin levels in freshly isolated and cultured human scalp hair follicle cells.

Abstract: Calmodulin levels were measured by radioimmuno-assay in freshly isolated and cultured psoriatic human scalp hair follicle cells. The mean value ± SEM for calmodulin was 1.97±0.15 ng calmodulin μg-1 protein for 16 control subjects whereas calmodulin levels were significantly increased in psoriatic hair follicles, 2.93±0.26 ng calmodulin μg-1 protein (uninvolved skin) for 18 patients and 3.09±0.21 ng calmodulin μg-1 protein for involved skin derived hair follicles for 17 of these patients. In vitro, 3-week-old cultures of psoriatic keratinocytes contained less DNA and more calmodulin per DNA than their normal counterparts. When 6 week-old cultures of psoriatic and control hair follicle keratinocytes were compared, this difference disappeared. These results are related to the state of differentiation of these cultures.

Isolation of plasma membranes from Drosophila embryos.

Abstract: Cell surface components probably play an important role in early embryonic development. However, hardly any information is available on the structure or regulation of expression of the corresponding genes. As a first step in approaching this issue, we devised a procedure to obtain enriched plasma membranes from embryonic Drosophila cells. Membranes are fractionated according to two independent physical parameters: size, using velocity gradient centrifugation and density, using isopicnic gradient centrifugation. The final membrane fraction is enriched by 6 to 8 fold with respect to the plasma membrane enzyme marker Na+/K+ ATPase and substantially depleted of the mitochondrial enzyme marker cytochrome C oxidase. Two-dimensional polyacrylamide gel electrophoresis of the purified membranes reveals enrichment for specific proteins and electron microscopy reveals membrane vesicles in abundance. The enriched fraction should be suitable for the preparation of antibody probes that recognize cell surface components.

Effects of different amino-group reagents on ribosomal integrity: structural role of lysine residues.

Abstract: Treatment of 60S subunits from yeast ribosomes with dicarboxylic acid anhydrides (maleic, dimethylmaleic and tetrahydrophtalic), which introduces negatively-charged residues, is accompanied by substantial dissociation of protein components (35–55%). In contrast, acetic anhydride or cyanate, which introduce uncharged groups, cause practically no protein release, even after extensive modification. Therefore, in addition to blocking lysine-RNA interactions, a large change in the electric charge of the proteins appears to be necessary to obtain dissociation. These results seem to indicate that lysine residues are not essential to ribosome integrity, while arginine-RNA interactions should play an important role in the maintenance of ribosomal structure.

Developmental changes in the chromatin of the brain of the rat: analysis by nicktranslation

Abstract: Nick translation of nuclei of the brain of 3- 14- and 30-day old rats was carried out following their digestion by DNase I. The incorporation of 3H-dTMP at 14- and 30-day is significantly lower than at 3-day. This may be due to a lower proportion of active chromatin (DNase I hypersensitive sites) and condensation of chromatin with progressive development. When nuclei were digested by EcoRI and then nick-translated, the incorporation of 3H-dTMP showed the same pattern. Since the EcoRI sites are believed to be randomly distributed, the overall conformation of chromatin including the DNase I sensitive sites seems to undergo increasing compaction with development.

In vitro ADP-ribosylation of chromosomal proteins of the brain of developing rats.

Abstract: In vitro ADP-ribosylation of chromosomal protein and its modulation by spermine, 3-aminobenzamide (3-AB) and benzamide were studied by incubating the nuclei of cerebral hemisphere of 3-, 14- and 30-day old rats with 32P-NAD+. Histones get ADP-ribosylated more than the non-histone chromosomal (NHC) protein. H1 is the major target for ADP-ribosylation. Among the nucleosomal histones, H2B is ADP-ribosylated most. The other core histones also get ADP-ribosylated to a lesser extent. ADP-ribosylation of both histones and NHC proteins decreases during development. Spermine stimulates, whereas 3-AB and benzamide inhibit, 32P-ADP-ribose incorporation into histones and NHC proteins. These effects decrease with development. Mild digestion of chromatin by micrococcal nuclease (MNase), EcoRI, and AluI prior to ADP-ribosylation stimulates incorporation of 32P-ADP-ribose. The degree of stimulation decreases as development proceeds. Such alterations indicate progressive condensation of chromatin with development.

Nuclear matrix proteins of Physarum polycephalum

Abstract: The proteins of nuclear matrix preparations from Physarum polycephalum were compared with analogous mammalian fractions by gel electrophoresis, DNA-binding studies and immunological tests. Polypeptides of 28 and 36 K dalton, which dominate in Physarum preparations, differed from calf thymus matrix proteins in that they were basic and showed low affinity to DNA. These polypeptides were present at about 1.2 mg per mg of nuclear DNA. Polypeptides of higher molecular weight occurred in the preparation at about 0.5 mg per mg of nuclear DNA. At least some of the latter proteins showed high affinity to DNA and cross-reacted with the antiserum against calf thymus matrix proteins.

DNA degradation after interaction of cis- and trans-diamminedichloroplatinum (II) with calf thymus nuclei.

Abstract: DNA samples isolated from control nuclei and nuclei treated by cis- and trans-diamminedichloroplatinum (DDP) were analyzed by gel electrophoresis. There were no changes in Mr when DNA isolated from nuclei treated with trans-DDP was analyzed. Scans of DNA isolated from nuclei treated with cis-DDP revealed significant changes in Mr. This DNA bears, however, no signs of regular fragmentation. The possible involvement of endonuclease activity in the degradation process is discussed.

Specific binding affinity for DNA of the L phage (Salmonella typhimurium) in extracts of Escherichia coli.

Abstract: The repressor gene cII of the L phage was cloned into plasmid pHC624 and expressed in E. coli. Two separate binding affinities for L phage DNA were identified during fractionation of protein extract of that strain. The activity that salts out in low concentration of ammonium sulphate belonged to the repressor, the activity that salts out in high concentrations of (NH4)2SO4 was proved to be of E. coli origin. Binding sites for the two proteins are located on different fragments of the L phage genome.

Modification of 40S ribosomal subunits from yeast with dimethylmaleic anhydride

Abstract: Modification of 40S ribosomal subunits from Saccharomyces cerevisiae with dimethylmaleic anhydride (DMMA1), a reagent for protein amino groups, is accompanied by loss of polypeptide-synthesizing activity and by dissociation of proteins from the particles. The protein-deficient ribosomal particles, originated from 40S subunits by treatment with dimethylmaleic anhydride at a molar ratio of reagent to particle of 250, can partially reconstitute active subunits upon addition of the corresponding released proteins, and regeneration of the modified amino groups.