Binding of the S7 Protein with Fragment 926–986/1219–1393 of the 16S rRNA As a Key Step in the Assembly of the Small Subunit of Prokaryotic Ribosomes

Both structural and thermodynamic studies are necessary to understand the ribosome assembly. An initial step was made in studying the interaction between a 16S rRNA fragment and S7, a key protein in assembling the prokaryotic ribosome small subunit. The apparent dissociation constant was obtained for complexes of recombinant Escherichia coliandThermus thermophilusS7 with a fragment of the 3" domain of the E. coli16S rRNA. Both proteins showed high rRNA-binding activity, which was not observed earlier. Since RNA and proteins are conformationally labile, their folding must be considered to correctly describe the RNA–protein interactions.     

Sequence organization of the chloroplast ribosomal spacer ofSpinacia oleracea including the 3′ end of the 16S rRNA and the 5′ end of the 23S rRNA

The 2201-bp spacer between the chloroplast ribosomal 16S and 23S genes ofSpinacia oleracea was sequenced. It contains the genes of the tRNA^Ile (GAU) and tRNA^Ala (UGC) which are both interrupted by introns of respectively 728 and 816 bp. These introns belong to the class II according to the classfication of Michel and Dujon [17]. Comparison of the rDNA spacer sequence of maize, tobacco and spinach indicates that no conserved polypeptide is encoded within the introns of the two tRNA genes and that the two main insertions/deletions between the three plants are located within two loops of the class II introns secondary structure, which is therefore conserved. Based on the sequence complementarity observed between the upstream and downstream parts, of the 16S and 23S rRNA genes, RNase III-like secondary structures involved in the processing of the rRNA precursor are proposed.     

Arrangement of the Template 3′ of the A-Site Codon on the Human 80S Ribosome

The arrangement of the template sequence 3′ of the A-site codon on the 80S ribosome was studied using mRNA analogs containing Phe codon UUU at the 5′ end and a photoreactive perfluoroarylazido group linked to C5 of U or N7 of G. The analogs were positioned on the ribosome with the use of tRNA^Phe, which directed the UUU codon to the P site, bringing a modified nucleotide to position +9 or +12 relative to the first nucleotide of the P-site codon. Upon mild UV irradiation of ribosome complexes, the analogs of both types crosslinked to the 18S rRNA and proteins of the 40S subunit. Comparisons were made with the crosslinking patterns of complexes in which an mRNA analog contained a modified nucleotide in position +7 (the crosslinking to 18S rRNA in such complexes has been studied previously). The efficiency of crosslinking to ribosomal components depended on the nature of the modified nucleotide of an mRNA analog and its position on the ribosome. The extent of crosslinking to the 18S rRNA drastically decreased as the modified nucleotide was transferred from position +7 to position +12. The 18S rRNA nucleotides involved in crosslinking were identified. A modified nucleotide in position +9 crosslinked to the invariant dinucleotide A1824/A1825 and variable A1823 in the 3′ minidomain of the 18S rRNA and to S15. The same ribosomal components have earlier been shown to crosslink to modified nucleotides in positions +4 to +7. In addition, all mRNA analogs crosslinked to invariant C1698 in the 3′ minidomain and to conserved region 605–620, which closes helix 18 in the 5′ domain.     

Age-dependent Expression of S100β in the Brain of Mice

S100β is a soluble calcium binding protein released by glial cells. It has been reported as a neurotrophic factor that promotes neurite maturation and outgrowth during development. This protein also plays a role in axonal stability and in long term potentiation in the adult brain. The ability of S100β to modulate neuronal morphology raises the important question whether there is an age-related difference in the expression of S100β in the cerebral and cerebellar cortices of AKR strain mice and is this change is region specific. Our RT–PCR and Western blotting experiments show that the expression of S100β gene in the cerebral and cerebellar cortices starts from 0 day, peaks at about 45 days. However, in 70-week old mice its expression is significantly up-regulated as compared to that of 20-week old mice. S100β follows the same age-related pattern in both cerebral and cerebellar cortices. These results suggest that S100β is important for brain development and establishment of proper brain functions. Up-regulation of S100β in old age may have some role in development of age-related pathological systems in the brain.     

Phytoplankton biomass in the sub-Antarctic area of the Straits of Magellan (53°S), Chile during spring-summer 1997/1998

In order to provide a better understanding of the dynamics of phytoplankton in the coastal regions of high latitudes, a study was carried out to estimate the dynamics of carbon biomass of autotrophic and heterotrophic algal groups over the austral spring-summer 1997/1998 period. At a fixed station located in the central basin (Paso Ancho) of the Straits of Magellan (53°S), surface water samples were collected at least once a week from September 1997 (early spring) to March 1998 (late summer). Quantitative analysis of biomass of phytoplankton was estimated from geometric volumes, using non-linear equations, and converted to biomass. The pattern of chlorophyll a showed a strong temporal variability, with maximum values (mean 2.8 mg m⁻³) at the austral spring phytoplankton increase or bloom (October/November) and minimum values during early spring (September: <0.5 mg m⁻³) and summer (January/March: 0.5–1.0 mg m⁻³). During the spring bloom, diatoms made up to 90% of the total phytoplankton carbon (0.01–189 μg l⁻¹), followed by a maximum of thecate dinoflagellates (0.08–34 μg l⁻¹), and sporadic high biomass of phytoflagellates during summer. Heterotrophic algal groups such as Gymnodinium and Gyrodinium spp. dominated (70%, in the 5- to 25-μm size range) shortly before the main diatom bloom, and small peaks were observed within spring and early summer periods (0–0.4 μg l⁻¹). Phytoflagellates dominated earlier (spring) with higher carbon biomass (8 μg l⁻¹) and post-bloom periods (summer) when carbon biomass ranged between 1 and 4 μg l⁻¹. Accepted: 6 September 2000     

Protein S3 in the human 80S ribosome adjoins mRNA 3′ of the A-site codon

The protein environment of mRNA 3′ of the A-site codon (the decoding site) in the human 80S ribosome was studied using a set of oligoribonucleotide derivatives bearing a UUU triplet at the 5′-end and a perfluoroarylazide group at one of the nucleotide residues 3′ of this triplet. Analogues of mRNA were phased into the ribosome using binding at the tRNA^Phe P-site, which recognizes the UUU codon. Mild UV irradiation of ribosome complexes with tRNA^Phe and mRNA analogues resulted in the predominant crosslinking of the analogues with the 40S subunit components, mainly with proteins and, to a lesser extent, with rRNA. Among the 40S subunit ribosomal proteins, the S3 protein was the main target for modification in all cases. In addition, minor crosslinking with the S2 protein was observed. The crosslinking with the S3 and S2 proteins occurred both in ternary complexes and in the absence of tRNA. Within ternary complexes, crosslinking with S15 protein was also found, its efficiency considerably falling when the modified nucleotide was moved from positions +5 to +12 relative to the first codon nucleotide in the P-site. In some cases, crosslinking with the S30 protein was observed; it was most efficient for the derivative containing a photoreactive group at the +7 adenosine residue. The results indicate that the S3 protein in the human ribosome plays a key role in the formation of the mRNA binding site 3′ of the codon in the decoding site.     

Use of the STR loci D18S53, D18S59, and D18S488 in the diagnosis of Edwards’ syndrome

The aim of this study was to investigate the feasibility of using short tandem repeats (STRs) to diagnose Edwards’ syndrome (ES). Quantitative fluorescence polymerase chain reaction (QF-PCR) was performed to amplify STR loci on chromosome 18, specifically D18S53, D18S59, and D18S488. The amplified products were subjected to a fluorescence signal analysis and their application to ES diagnosis was examined. Among the 807 cases that showed normal results in the karyotype analysis, 793 showed one or two fluorescence bands with a fluorescence intensity ratio of 1:1, and 14 cases showed 3 bands, which were false-positive results. ES was diagnosed in 9 samples. The sensitivities of D18S53, D18S59, and D18S488 for the diagnosis of ES were 77.78, 44.44, and 55.56 % and the specificities were 96.16, 96.03, and 96.28 %, respectively. The combined sensitivity of the three loci for diagnosing DS was 100 % (9/9), with a specificity of 98.27 % (793/807). QF-PCR amplification of STR loci had high sensitivity, strong specificity, and was simple and rapid. Thus, it might have wide clinical applications, and could be an ideal tool for large-scale genetic and prenatal diagnosis of ES.     

The Cdc7/Dbf4 protein kinase: target of the S phase checkpoint?

Cdc7/Dbf4 is a protein kinase that is required for the initiation of DNA replication in eukaryotes. Recent work has provided new clues to the role that Cdc7/Dbf4 plays in this process. A range of other observations suggest that Cdc7/Dbf4 also plays another, less well characterized, role in checkpoint function and in the maintenance of genomic integrity. In this review we attempt to bring together new information to explain how Cdc7/Dbf4 may perform these two distinct functions.     

Nucleotide sequence of the 17S–25S spacer region from rice rDNA

Summary The nucleotide sequence of a spacer region between rice 17S and 25S rRNA genes (rDNAs) has been determined. The coding regions for the mature 17S, 5.8S and 25S rRNAs were identified by sequencing terminal regions of these rRNAs. The first internal transcribed spacer (ITS1), between 17S and 5.8S rDNAs, is 194–195 bp long. The second internal transcribed spacer (ITS2), between 5.8S and 25S rDNAs, is 233 bp long. Both spacers are very rich in G+C, 72.7% for ITS1 and 77.3% for ITS2. The 5.8S rDNA is 163–164 bp long and similar in primary and secondary structures to other eukaryotic 5.8S rDNAs. The 5.8S rDNA is capable of interacting with the 5′ terminal region of 25S rDNA.     

Functional assembly of the λ S holin requires periplasmic localization of its N-terminus

Bacteriophage-λ-induced host-cell lysis requires two phage-encoded proteins, the S holin and the R transglycosylase. At a specific time during infection, the holin forms a lesion in the cytoplasmic membrane that permits access of the R protein to its substrate, the peptidoglycan. The λS gene represents the prototype of holin genes with a dual-start motif; they encode two proteins, a lysis effector and a lysis inhibitor. Although the two S proteins differ only by two amino acids (Met-1 and Lys-2) at the N-terminus, the longer product (S107) acts as an inhibitor of the lysis effector (S105). The functional difference between the proteins has been previously ascribed to the Lys-2 residue in S107. It was therefore of interest to determine the subcellular localization of the N-terminus of either S protein. To study the membrane topology of the S proteins, we used the topology probe TEM β-lactamase and an N-terminal tag derived from the Pseudomonas aeruginosa phage Pf3 coat protein. We show that both S proteins have a type III (N_out/C_in) topology. The results provide insight into the regulatory mechanism imposed by the dual-start motif and will be discussed in terms of a model for temporal regulation of the S-dependent “hole” in the membrane. Received: 28 January 1999 / Accepted: 23 April 1999     

Impact of polymorphism of the regulatory subunit of the ��-calpain (CAPN1S) on the proteolysis process and meat tenderness of young cattle

The objective of this study was to estimate the impact of the polymorphism of ??-calpain (CAPN1S) gene on protein changes of the cattle muscle tissue and its tenderness during 10-day cold storage. The analysis was performed on the longest dorsal and lumbar muscles collected from 76 bulls 6 to 12 months of age. Polymorphism identification of the above-mentioned gene was conducted using the PCR-RFLP technique. Its effect on the course of the proteolysis process was assessed by monitoring changes in proportions of tissue proteins during 10-day process of meat ageing. Special attention was focused on changes in native titin (T1) share and products of its degradation (proteins of molecular weight (m.w.) of 2400 and 200 kDa), ??-actinin and protein of 37 kDa as well as myosin heavy chains (MHC). In the case of the last proteins, their polymorphism was evaluated as well. Meat tenderness was estimated measuring the value of shear force and sensorially. The highest tenderness was ascertained for the heterozygote. Its improvement was associated with a significant decrease in proportions of proteins of molecular weight of approximately 37 kDa accompanied by an increase of those with 200 kDa molecular weight. Muscles derived from cattle of CT genotype were characterised by the highest proportions of type 2a MHC isoform. Value differences between proportions determined for the heterozygote and CC and TT homozygotes of the CAPN1S gene were statistically significant. Therefore, it can be presumed that the process of meat tenderisation was especially connected with MHC polymorphism.     

Sequence Diversity in the 16S–23S Intergenic Spacer Region (ISR) of the rRNA Operons in Representatives of the Escherichia coli ECOR Collection

The ribosomal RNA multigene family in Escherichia coli comprises seven rrn operons of similar, but not identical, sequence. Four operons (rrnC, B, G, and E) contain genes in the 16S–23S intergenic spacer region (ISR) for tRNA^Glu-2 and three (rrnA, D, and H) contain genes for tRNA^Ile-1 and tRNA^Ala-1B. To increase our understanding of their molecular evolution, we have determined the ISR sequence of the seven operons in a set of 12 strains from the ECOR collection. Each operon was specifically amplified using polymerase chain reaction primers designed from genes or open reading frames located upstream of the 16S rRNA genes in E. coli K12. With a single exception (ECOR 40), ISRs containing one or two tRNA genes were found at the same respective loci as those of strain K12. Intercistronic heterogeneity already found in K12 was representative of most variation among the strains studied and the location of polymorphic sites was the same. Dispersed nucleotide substitutions were very few but 21 variable sites were found grouped in a stem-loop, although the secondary structure was conserved. Some regions were found in which a stretch of nucleotides was substituted in block by one alternative, apparently unrelated, sequence (as illustrated by the known putative insertion of rsl in K12). Except for substitutions of different sizes and insertions/deletions found in the ISR, the pattern of nucleotide variation is very similar to that found for the 16S rRNA gene in E. coli. Strains K12 and ECOR 40 showed the highest intercistronic heterogeneity. Most strains showed a strong tendency to homogenization. Concerted evolution could explain the notorious conservation of this region that is supposed to have low functional restrictions. Received: 31 July 1997 / Accepted: 17 October 1997     

The Rieske Fe/S protein of the cytochrome b6/f complex in chloroplasts: missing link in the evolution of protein transport pathways in chloroplasts?

The Rieske Fe/S protein, a nuclear-encoded subunit of the cytochrome b(6)/f complex in chloroplasts, is retarded in the stromal space after import into the chloroplast and only slowly translocated further into the thylakoid membrane system. As shown by the sensitivity to nigericin and to specific competitor proteins, thylakoid transport takes place by the DeltapH-dependent TAT pathway. The Rieske protein is an untypical TAT substrate, however. It is only the second integral membrane protein shown to utilize this pathway, and it is the first authentic substrate without a cleavable signal peptide. Transport is instead mediated by the NH(2)-terminal membrane anchor, which lacks, however, the twin-arginine motif indicative of DeltapH/TAT-dependent transport signals. Furthermore, transport is affected by sodium azide as well as by competitor proteins for the Sec pathway in chloroplasts, demonstrating for the first time some cross-talk of the two pathways. This might take place in the stroma where the Rieske protein accumulates after import in several complexes of high molecular mass, among which the cpn60 complex is the most prominent. These untypical features suggest that the Rieske protein represents an intermediate or early state in the evolution of the thylakoidal protein transport pathways.