Structural Relationships Among the Ribosomal Stalk Proteins from the Three Domains of Life

The GTPase center of the large ribosomal subunit, being a landing platform for translation factors, and regarded as one of the oldest structures in the ribosome, is a universally conserved structure in all domains of life. It is thought that this structure could be responsible for the major breakthrough on the way to the RNA/protein world, because its appearance would have dramatically increased the rate and accuracy of protein synthesis. The major part of this center is recognized as a distinct structural entity, called the stalk. The main functional part of the stalk in all domains of life is composed of small L12/P proteins, which are believed to form an evolutionarily conserved group. However, some data indicate that the bacterial and archaeo/eukaryal proteins are not related to each other structurally, and only a functional relationship may be clearly recognized. To clarify this point, we performed a comprehensive comparative analysis of the L12/P proteins from the three domains of life. The results show that bacterial and archaeo/eukaryal L12/P-proteins are not structurally related and, therefore, might not be linked evolutionarily either. Consequently, these proteins should be regarded as analogous rather than homologous systems and probably appeared on the ribosomal particle in two independent events in the course of evolution.     

Cerebrospinal Fluid Proteins Studied by Two-Dimensional Gel Electrophoresis and Immunoblotting Technique

The proteins of lumbar CSF have been investigated by two-dimensional gel electrophoresis, and their patterns have been compared with the corresponding serum protein patterns. Serum proteins in CSF have been identified by electroblotting and immunoreaction with antiserum against total human serum proteins. Proteins derived from brain have been identified with antiserum against human brain proteins. The most prominent CSF protein group has been identified as a multiple form of apolipoprotein E. The correct position of the glial fibrillary acidic protein has also been determined. The prefractionation of CSF proteins by size exclusion chromatography or by affinity chromatography followed by two-dimensional electrophoresis has facilitated the detection of trace components in CSF and the corresponding serum.     

Heat Shock Effects on the Circadian Rhythm of Protein Synthesis and Phosphorylation of Ribosomal Proteins in Gonyaulax polyedra

Circadian changes in protein synthesis and phosphorylation of ribosomal and cytoplasmic proteins in the marine dinoflagellate Gonyaulax polyedra were analyzed by radioactive labeling and polyacrylamide gel electrophoresis. Maximal rates of protein synthesis were found during the subjective night and minimal rates during the subjective day. Protein synthesis was inhibited by heat shock to a different extent at different circadian phases—maximally during the subjective night. Heat shock proteins (HSPs) having molecular weights of approximately 105, 89, 83, 66, 35, and 18 kDa were induced by these treatments. Induction of HSP89 and HSP35 showed circadian differences with maximal synthesis rates at CT 15, whereas most HSPs maintained a constant constitutive and induced synthesis. Recovery of normal protein synthesis after heat shock occurred faster during the subjective night than during the subjective day. Ribosomal proteins with molecular weights of 16 and 18 kDa were highly phosphorylated by [³⁵S] thio gamma adenosine triphosphate during day phase in a light-dark cycle or at CT 6 in constant dim light and labeled only to a minor degree during night phase or at CT 18. A ribosome-associated protein (35 kDa) was labeled during the day and not during the night, but after heat shock during both day and night. In the 200,000 g cytosolic fraction, a 35-kDa protein was found to be more intensely labeled at night than during the day phase after heat shock. The results of this study show a correlation between circadian changes in the overall protein synthesis and ribosomal protein phosphorylation. The rhythm of protein synthesis and phosphorylation of a ribosome-associated protein are drastically altered by heat shock and dependent on the circadian phase.     

Archaebacteria

Summary Experimental work published elsewhere has shown that the Archaebacteria encompass several distinct subgroups including methanogens, extreme halophiles, and various thermoacidophiles. The common chacteristics of Archaebacteria known to date are these: (1) the presence of characteristic tRNAs and ribosomal RNAs; (2) the absence of peptidogly can cell walls, with in many cases, replacement by a largely proteinaceous coat; (3) the occurrence of ether linked lipids built from phytanyl chains and (4) in all cases known so far, their occurrence only in unusual habitats.These organisms contain a number of eucaryotic features in addition to their many bacterial attributes. This is interpreted as a strong indication that the Archaebacteria, while not actually eucaryotic, do indeed represent a third separate, line of descent as originally proposed.     

Molecular phylogenies based on ribosomal protein L11, L1, L10, and L12 sequences

Summary Available sequences that correspond to the E. coli ribosomal proteins L11, L1, L10, and L12 from eubacteria, archaebacteria, and eukaryotes have been aligned. The alignments were analyzed qualitatively for shared structural features and for conservation of deletions or insertions. The alignments were further subjected to quantitative phylogenetic analysis, and the amino acid identity between selected pairs of sequences was calculated. In general, eubacteria, archaebacteria, and eukaryotes each form coherent and well-resolved nonoverlapping phylogenetic domains. The degree of diversity of the four proteins between the three groups is not uniform. For L11, the eubacterial and archaebacterial proteins are very similar whereas the eukaryotic L11 is clearly less similar. In contrast, in the case of the L12 proteins and to a lesser extent the L10 proteins, the archaebacterial and eukaryotic proteins are similar whereas the eubacterial proteins are different. The eukaryotic L1 equivalent protein has yet to be identified. If the root of the universal tree is near or within the eubacterial domain, our ribosomal protein-based phylogenies indicate that archaebacteria are monophyletic. The eukaryotic lineage appears to originate either near or within the archaebacterial domain. Correspondence to: P. Dennis     

枯草杆菌核糖体蛋白质突变对碱性蛋白酶基因表达的影响

用枯草杆菌体外转录-翻译偶联系统检测13种19株枯草杆菌核糖体蛋白质突变对碱性蛋白酶基因表达的影响,发现10种13株核糖体蛋白质突变能影响碱性蛋白酶基因的表达。其中依赖链霉素突变核糖体几乎不能翻译碱性蛋白酶mRNA。依赖链霉素突变在翻译层次抑制碱性蛋白酶基因的表达,但对中性蛋白酶基因的表达没有影响。在碱性蛋白酶mRNA翻译起始区有一个复合二级结构,用体外突变方法破坏其中一个,翻译效率提高8.2倍。依赖链霉素突变和抗链霉素突变核糖体的高级结构不同,与碱性蛋白酶mRNA 5'端片段的亲合力也有差异。由于碱性蛋白酶mRNA翻译起始区的复合二级结构和低起始强度以及依赖链霉素突变核糖体高级结构的改变,使依赖链霉素突变核糖体不能翻译碱性蛋白酶mRNA。     

Horizontal gene flow from Eubacteria to Archaebacteria and what it means for our understanding of eukaryogenesis

The origin of the eukaryotic cell is considered one of the major evolutionary transitions in the history of life. Current evidence strongly supports a scenario of eukaryotic origin in which two prokaryotes, an archaebacterial host and an α-proteobacterium (the free-living ancestor of the mitochondrion), entered a stable symbiotic relationship. The establishment of this relationship was associated with a process of chimerization, whereby a large number of genes from the α-proteobacterial symbiont were transferred to the host nucleus. A general framework allowing the conceptualization of eukaryogenesis from a genomic perspective has long been lacking. Recent studies suggest that the origins of several archaebacterial phyla were coincident with massive imports of eubacterial genes. Although this does not indicate that these phyla originated through the same process that led to the origin of Eukaryota, it suggests that Archaebacteria might have had a general propensity to integrate into their genomes large amounts of eubacterial DNA. We suggest that this propensity provides a framework in which eukaryogenesis can be understood and studied in the light of archaebacterial ecology. We applied a recently developed supertree method to a genomic dataset composed of 392 eubacterial and 51 archaebacterial genera to test whether large numbers of genes flowing from Eubacteria are indeed coincident with the origin of major archaebacterial clades. In addition, we identified two potential large-scale transfers of uncertain directionality at the base of the archaebacterial tree. Our results are consistent with previous findings and seem to indicate that eubacterial gene imports (particularly from δ-Proteobacteria, Clostridia and Actinobacteria) were an important factor in archaebacterial history. Archaebacteria seem to have long relied on Eubacteria as a source of genetic diversity, and while the precise mechanism that allowed these imports is unknown, we suggest that our results support the view that processes comparable to those through which eukaryotes emerged might have been common in archaebacterial history.     

多肽抗体谱免疫印迹法检测ENA抗体的临床应用

本文报道应用多肽抗体谱印迹法检测风湿性疾病患者的ＥＮＡ抗体,该法一次可检测７种抗体。应用本法检测ＳＬＥ８５例,其Ｓｍ抗体阳性率为２０．０％,ｕ１－ＲＮＰ抗体为４１．１％,ＳＳ－Ａ抗体为１０．５％ＳＳ－Ｂ抗体为３．５％,抗核糖体为７．０％。检测ＤＬＥ６例,其山ｕ１－ＲＮＰ抗体阳性率为３３．０％。检测ＰＭ／ＤＭ１５例,其ｕ１－ＲＮＰ抗体阳性率为１３．０％,Ｊｏ－１抗体为１４．０％。检测ＲＡ４８例,其ｕ１－ＲＮＰ抗体的阳性率为３３．３％。检测ＳＳ２１例,其ＳＳ－Ａ抗体阳性率为４３．０％,ＳＳ－Ｂ抗体为３８．０％,检测ＰＳＳ９例,其Ｓｃｌ－７０抗体阳性率为２２．０％。检测ＭＣＴＤ１０例,其ｕ１－ＲＮＰ抗体阳性率为５０．０％。健康人对照１００例,７种抗体均为阴性。显示应用多肽抗体谱印迹法检测结果与国内其它方法所测得结果基本一致,但可检测的抗体种类多于其它方法。     

Charge Segregation and Low Hydrophobicity Are Key Features of Ribosomal Proteins from Different Organisms

Ribosomes are large and highly charged macromolecular complexes consisting of RNA and proteins. Here, we address the electrostatic and nonpolar properties of ribosomal proteins that are important for ribosome assembly and interaction with other cellular components and may influence protein folding on the ribosome. We examined 50 S ribosomal subunits from 10 species and found a clear distinction between the net charge of ribosomal proteins from halophilic and non-halophilic organisms. We found that ∼67% ribosomal proteins from halophiles are negatively charged, whereas only up to ∼15% of ribosomal proteins from non-halophiles share this property. Conversely, hydrophobicity tends to be lower for ribosomal proteins from halophiles than for the corresponding proteins from non-halophiles. Importantly, the surface electrostatic potential of ribosomal proteins from all organisms, especially halophiles, has distinct positive and negative regions across all the examined species. Positively and negatively charged residues of ribosomal proteins tend to be clustered in buried and solvent-exposed regions, respectively. Hence, the majority of ribosomal proteins is characterized by a significant degree of intramolecular charge segregation, regardless of the organism of origin. This key property enables the ribosome to accommodate proteins within its complex scaffold regardless of their overall net charge.     

Identification of a eukaryotic-like protein kinase gene in Archaebacteria.

Primary sequence patterns based on known conserved sites in eukaryotic protein kinases were used to search for eukaryotic-like protein kinase sequences in a six-frame translation of the bacterial subsection of GenBank. This search identified a previously unrecognized eukaryotic-like protein kinase gene in three related methanogenic archaebacteria, Methanococcus vannielii, M. voltae, and M. thermolithotrophicus. The proposed coding sequences are located in orthologous open reading frames (ORFs): ORF547, ORF294, and ORF114, respectively. The C-terminus of the ORFs contains 9 of the 11 subdomains characteristically conserved within the eukaryotic protein kinase catalytic domain. The N-terminus of the ORFs is similar to a putative glycoprotease in Pasteurella haemolytica and its homologue in Escherichia coli, the orfX gene. This is the first report of a eukaryotic-like protein kinase sequence observed in Archaebacteria.     

Escherichia coli 核糖体蛋白质突变影响Lambda噬菌体N基因的表达

λ Nam 7除其cI基因是cI 857温度敏感突变外,其N基因携有amber无义突变,故在Eseherichia coli A19(met,thi,his-95,rna-19,rel-1)上不能增殖。λcI 857只有cI 857温度敏感突变,其N基因是野生型,所以能以E. coli A19为宿主进行增殖。λcI 857和λ Nam7只有1个N基因之差。λcI 857在A19及其衍生株上增殖的优劣,可以作为判断N基因表达程度高低的标准。本文以24种核糖体蛋白质突变体为宿主,测定λcI 857的成斑率。结果在S3+L22,S4+L16+L24,S21+L25,L24,缺L1,缺S3等突变体中,成斑率下降到10~(-6)—10~(-6);在S3+S18+L6+L24+L27和L27突变体中,成斑率分别提高6.02和3.56倍。以上结果说明核糖体蛋白质突变影响λ N基因的表达。     

核糖体蛋白参与调控p53诱导活化的分子机制

核转录因子p53是重要的肿瘤抑制因子,具有DNA损伤修复、促细胞凋亡、促细胞分化及增殖抑制等功能,并通过调控细胞周期行进和促进细胞凋亡发挥肿瘤抑制功能。原癌蛋白MDM2为p53的E3泛素化连接酶,MDM2－p53信号轴的功能异常与多种恶性肿瘤的发生发展相关。核糖体蛋白（RP）是蛋白质合成反应的关键调节蛋白,其功能失常与多种疾病相关。近年来的研究发现,RP能通过调节MDM2－p53信号轴在p53相关性肿瘤调控中发挥重要作用。我们根据目前的研究进展,对RP－MDM2－D53信号轴进行简要综述。     

Phylogenetic Relationships of Fungi, Plantae, and Animalia Inferred from Homologous Comparison of Ribosomal Proteins

The complete set of available ribosomal proteins was utilized, at both the peptidic and the nucleotidic level, to establish that plants and metazoans form two sister clades relative to fungi. Different phylogenetic inference methods are applied to the sequence data, using archeans as the outgroup. The evolutionary length of the internal branch within the eukaryotic crown trichotomy is demonstrated to be, at most, one-tenth of the evolutionary length of the branch leading to the cenancester of these three kingdoms. Received: 1 November 1997 / Accepted: 7 January 1998     

Axonal Polypeptides Cross-Reactive with Antibodies to Neurofilament Proteins

Antibodies were prepared to mammalian CNS neurofilament proteins (NFPs) and the antibody specificities were compared using a sensitive immunoblotting method. This procedure was used to detect and characterize cross-reactive proteins and their degradation products in neurofilament preparations. NFPs were prepared by axon flotation. Rabbits were immunized with 200,000, 140,000, and 70,000 NFPs (200K, 140K, and 70K) that had been electrophoretically purified by polyacrylamide gel electrophoresis (PAGE). By immunohistofluorescence it was shown that all antisera stained similar filamentous structures in rat cerebellar neurons. By use of a horseradish peroxidase-conjugated indirect antibody procedure, however, differences were detected in the cross-reactivities of the antisera to rat NFPs, separated by PAGE and electrophoretically transferred to nitrocellulose membranes. Each antiserum exhibited strong binding to the homologous NFP and, thus, was suitable for the detection of cross-reactive polypeptides and proteolytic degradation products derived exclusively from the individual NFPs. Anti-200K, anti-140K, or anti-70K was applied to overloaded two-dimensional nitrocellulose blots of NFPs prepared by axon flotation. Each of the three sera detected a group of unique nonoverlapping polypeptides, some of which were identified as NFP degradation products. A different group of polypeptides was cross-reactive with antiserum to purified glial fibrillary acidic protein. The immunostaining of polypeptides on nitrocellulose was far more sensitive for detecting NFP degradation products than was staining polyacrylamide gels with Coomassie blue. Titers for the antisera were two to three orders of magnitude higher with the immunoblotting procedure than with immunohistologic methods. The sensitivity and the specificity of the described methods suggest their usefulness for examining proteolytic cleavage products of NFPs under a variety of conditions.