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A chemical interference study on the interaction of ribosomal protein L11 from Escherichia coli with RNA molecules containing its binding site from 23S rRNA.

The interaction between ribosomal protein L11 from Escherichia coli and in vitro synthesized RNA containing its binding site from 23S rRNA was characterized by identifying nucleotides that interfered with complex formation when chemically modified by diethylpyrocarbonate or hydrazine. Chemically modified RNA was incubated with L11 under conditions appropriate for specific binding of L11 and the resulting protein-RNA complex was separated from unbound RNA on Mg(2+)-containing polyacrylamide gels. The ability to isolate L11 complexes on such gels was affected by the extent of modification by either reagent. Protein-bound and free RNAs were recovered and treated with aniline to identify their content of modified bases. Exclusion of RNA containing chemically altered bases from L11-associated material occurred for 29 modified nucleotides, located throughout the region corresponding to residues 1055-1105 in 23S rRNA. Ten bases within this region did not reproducibly inhibit binding when modified. Multiple bands of RNA were consistently observed on the nondenaturing gels, suggesting that significant intermolecular RNA-RNA interactions had occurred.

Aniline Compounds↗

Impairment of RNA synthesis and its recovery in angelicin photosensitized mammalian cells. A probe for DNA damage and repair.

The template activity of DNA for ribosomal RNA transcription has been investigated in monkey kidney CV-1 cells after angelicin photosensitization in order to monitor the induction of lesions in the DNA and their possible subsequent disappearance, i.e. repair. Separate confluent cultures were submitted to a single angelicin treatment at different time intervals before incubation with [3H]uridine. The labeled RNA prepared from whole cells was analysed by polyacrylamide-agarose gel electrophoresis. The results indicate that: Angelicin monoadditions on DNA constitute transcription-terminating lesions which depress overall RNA synthesis, give rise to shortened RNA chains and modify the expression of transcriptional linked genes. CV-1 cells are able to repair, at least partially, the induced transcription-terminating lesions and progressively recover RNA synthesis with a reversion of the initially observed modifications. The repair seems to be independent of semiconservative DNA synthesis since fluorodeoxyuridine does not affect the recovery of RNA transcription. The present work also confirms the arrangement of rRNA genes in tandem behind a common operator in the order 18--28 S as previously determined in the same cells by a radiological mapping technique and reinforces the potential applicability of transcription analysis to the study of repair processes operating on physically or chemically induced damage in DNA.

Cell Line↗

Conventional and nonconventional roles of the nucleolus.

As the most prominent of subnuclear structures, the nucleolus has a well-established role in ribosomal subunit assembly. Additional nucleolar functions, not related to ribosome biogenesis, have been discovered within the last decade. Built around multiple copies of the genes for preribosomal RNA (rDNA), nucleolar structure is largely dependent on the process of ribosome assembly. The nucleolus is disassembled during mitosis at which time preribosomal RNA transcription and processing are suppressed; it is reassembled at the end of mitosis in part from components preserved from the previous cell cycle. Expression of preribosomal RNA (pre-rRNA) is regulated by the silencing of individual rDNA genes via alterations in chromatin structure or by controlling RNA polymerase I initiation complex formation. Preribosomal RNA processing and posttranscriptional modifications are guided by a multitude of small nucleolar RNAs. Nearly completed ribosomal subunits are exported to the cytoplasm by an established nuclear export system with the aid of specialized adapter molecules. Some preribosomal and nucleolar components are transiently localized in Cajal bodies, presumably for modification or assembly. The nonconventional functions of nucleolus include roles in viral infections, nuclear export, sequestration of regulatory molecules, modification of small RNAs, RNP assembly, and control of aging, although some of these functions are not well established. Additional progress in defining the mechanisms of each step in ribosome biogenesis as well as clarification of the precise role of the nucleolus in nonconventional activities is expected in the next decade.

Active Transport, Cell Nucleus↗

The mitochondrial genome of the sea anemone Metridium senile (Cnidaria): introns, a paucity of tRNA genes, and a near-standard genetic code.

The circular, 17,443 nucleotide-pair mitochondrial (mt) DNA molecule of the sea anemone, Metridium senile (class Anthozoa, phylum Cnidaria) is presented. This molecule contains genes for 13 energy pathway proteins and two ribosomal (r) RNAs but, relative to other metazoan mtDNAs, has two unique features: only two transfer RNAs (tRNA(f-Met) and tRNA(Trp)) are encoded, and the cytochrome c oxidase subunit I (COI) and NADH dehydrogenase subunit 5 (ND5) genes each include a group I intron. The COI intron encodes a putative homing endonuclease, and the ND5 intron contains the molecule's ND1 and ND3 genes. Most of the unusual characteristics of other metazoan mtDNAs are not found in M. senile mtDNA: unorthodox translation initiation codons and partial translation termination codons are absent, the use of TGA to specify tryptophan is the only genetic code modification, and both encoded tRNAs have primary and secondary structures closely resembling those of standard tRNAs. Also, with regard to size and secondary structure potential, the mt-s-rRNA and mt-1-rRNA have the least deviation from Escherichia coli 16S and 23S rRNAs of all known metazoan mt-rRNAs. These observations indicate that most of the genetic variations previously reported in metazoan mtDNAs developed after Cnidaria diverged from the common ancestral line of all other Metazoa.

Amino Acid Sequence↗

RNA-Binding Characteristics of a Ribonucleoprotein from Spinach Chloroplast.

A chloroplast (nuclear-encoded) RNA-binding protein (28RNP) was previously purified from spinach (Spinacia oleracea). This 28RNP was found to be the major RNA-binding protein co-purified during the isolation scheme of 3[prime] end RNA-processing activity of several chloroplastic genes. To learn more about the possible involvement of 28RNP in the 3[prime] end RNA-processing event, we investigated the RNA-binding properties and the location of the protein in the chloroplast. We found that recombinant Escherichia coliexpressed 28RNP binds with apparently the same affinity to every chloroplastic 3[prime] end RNA that was analyzed, as well as to RNAs derived from the 5[prime] end or the coding region of some chloroplastic genes. Differences in the RNA-binding affinities for some chloroplastic 3[prime] end RNAs were observed when the recombinant 28RNP was compared with the "native" 28RNP in the chloroplast-soluble protein extract. In addition, we found that the 28RNP is not associated with either thylakoid-bound or soluble polysomes in which a great portion of the chloroplast rRNA and mRNA are localized. These results suggest that the native 28RNP binds specifically to certain RNA molecules in the chloroplast in which other components (possibly proteins) and/or posttranslational modifications are involved in determining RNA-binding specificity of the 28RNP.

Journal Article↗

The macrolide-lincosamide-streptogramin B resistance phenotypes characterized by using a specifically deleted, antibiotic-sensitive strain of Streptomyces lividans.

Genes conferring resistance to macrolide, lincosamide, and streptogramin B (MLS) antibiotics via ribosomal modification are widespread in bacteria, including clinical isolates and MLS-producing actinomycetes. Such erm-type genes encode enzymes that mono- or dimethylate residue A-2058 of 23S rRNA. The different phenotypes resulting from monomethylation (MLS-I phenotype, conferred by erm type I genes) or dimethylation (MLS-II phenotype due to erm type II genes) have been characterized by introducing tlrD or ermE, respectively, into an MLS-sensitive derivative of Streptomyces lividans TK21. This strain (designated OS456) was generated by specific replacement of the endogenous resistance genes lrm and mgt. The MLS-I phenotype is characterized by high-level resistance to lincomycin with only marginal resistance to macrolides such as chalcomycin or tylosin, whereas the MLS-II phenotype involves high-level resistance to all MLS drugs. Mono- and dimethylated ribosomes were introduced into a cell-free protein-synthesizing system prepared from S. lividans and compared with unmodified particles in their response to antibiotics. There was no simple correlation between the relative potencies of MLS drugs at the level of the target site (i.e., the ribosome) and their antibacterial activities expressed as MICs.

Anti-Bacterial Agents↗

Simultaneous fluorescence in situ hybridization of mRNA and rRNA in environmental bacteria.

We developed for Bacteria in environmental samples a sensitive and reliable mRNA fluorescence in situ hybridization (FISH) protocol that allows for simultaneous cell identification by rRNA FISH. Samples were carbethoxylated with diethylpyrocarbonate to inactivate intracellular RNases and pretreated with lysozyme and/or proteinase K at different concentrations. Optimizing the permeabilization of each type of sample proved to be a critical step in avoiding false-negative or false-positive results. The quality of probes as well as a stringent hybridization temperature were determined with expression clones. To increase the sensitivity of mRNA FISH, long ribonucleotide probes were labeled at a high density with cis-platinum-linked digoxigenin (DIG). The hybrid was immunocytochemically detected with an anti-DIG antibody labeled with horseradish peroxidase (HRP). Subsequently, the hybridization signal was amplified by catalyzed reporter deposition with fluorochrome-labeled tyramides. p-Iodophenylboronic acid and high concentrations of NaCl substantially enhanced the deposition of tyramides and thus increased the sensitivity of our approach. After inactivation of the antibody-delivered HRP, rRNA FISH was performed by following routine protocols. To show the broad applicability of our approach, mRNA of a key enzyme of aerobic methane oxidation, particulate methane monooxygenase (subunit A), was hybridized with different types of samples: pure cultures, symbionts of a hydrothermal vent bivalve, and even sediment, one of the most difficult sample types with which to perform successful FISH. By simultaneous mRNA FISH and rRNA FISH, single cells are identified and shown to express a particular gene. Our protocol is transferable to many different types of samples with the need for only minor modifications of fixation and permeabilization procedures.

Bacteria↗

Role of modified nucleosides of yeast tRNA(Phe) in ribosomal binding.

Naturally occurring nucleoside modifications are an intrinsic feature of transfer RNA (tRNA), and have been implicated in the efficiency, as well as accuracy-of codon recognition. The structural and functional contributions of the modified nucleosides in the yeast tRNA(Phe) anticodon domain were examined. Modified nucleosides were site-selectively incorporated, individually and in combinations, into the heptadecamer anticodon stem and loop domain, (ASL(Phe)). The stem modification, 5-methylcytidine, improved RNA thermal stability, but had a deleterious effect on ribosomal binding. In contrast, the loop modification, 1-methylguanosine, enhanced ribosome binding, but dramatically decreased thermal stability. With multiple modifications present, the global ASL stability was mostly the result of the individual contributions to the stem plus that to the loop. The effect of modification on ribosomal binding was not predictable from thermodynamic contributions or location in the stem or loop. With 4/5 modifications in the ASL, ribosomal binding was comparable to that of the unmodified ASL. Therefore, modifications of the yeast tRNA(Phe) anticodon domain may have more to do with accuracy of codon reading than with affinity of this tRNA for the ribosomal P-site. In addition, we have used the approach of site-selective incorporation of specific nucleoside modifications to identify 2'O-methylation of guanosine at wobble position 34 (Gm34) as being responsible for the characteristically enhanced chemical reactivity of C1400 in Escherichia coli 16S rRNA upon ribosomal footprinting of yeast tRNA(Phe). Thus, effective ribosome binding of tRNA(Phe) is a combination of anticodon stem stability and the correct architecture and dynamics of the anticodon loop. Correct tRNA binding to the ribosomal P-site probably includes interaction of Gm34 with 16S rRNA C1400.

Nucleosides↗

Ribosomal genes as early targets of cadmium-induced toxicity in Chironomus riparius larvae.

Cadmium is a widespread environmental pollutant that causes severe impacts in organisms. Although the effects of cadmium on aquatic insects have been studied in terms of their toxicity and changes in developmental parameters, little is known about its molecular and genetic effects. We have investigated the alterations in the pattern of gene expression provoked by acute exposure to cadmium in Chironomus riparius Mg. (Diptera, Chironomidae), a sentinel organism widely used in aquatic toxicity testing. The early cytotoxic effects were evaluated using immunocytochemistry and specific fluorescent probes in fourth instar larvae after 12 h of 10 mM cadmium treatments; under these conditions no significant effect on larvae mortality was detected until after 36 h of exposure. The changes in the pattern of gene expression were analysed by means of DNA/RNA hybrid antibodies in the polytene chromosomes from salivary gland cells. A decrease in the activity of the nucleolus is especially remarkable, accompanied by a significant reduction in size and the modification in nucleolar architecture, as shown by FISH. The inhibition of rDNA transcription was further evaluated by Northern blot analysis, which showed a marked decrease in the level of preribosomal rRNA (54% 45S 12 h). However, the BR genes, whose products are the giant polypeptides that constitute the silk-like secretion for constructing housing tubes, remain active. Simultaneously, decondensation and activation take place at some chromosomal regions, especially at the centromeres. The changes observed in the pattern of gene expression do not resemble those found after heat shock or other cell stressors. These data provide the first evidence that cadmium interacts with ribosomal genes and results in a drastic impairment of the functional activity of the nucleolus, an essential organelle for cellular survival. Therefore, the depletion of ribosomes would be a long-term effect of Cd-induced cellular damage. These findings may have important implications for understanding the adverse biological effects of cadmium and its toxic mechanism, as yet not clearly defined, and provide a sensitive biomarker of cadmium exposure.

Animals↗

Probing the secondary structure of expansion segment ES6 in 18S ribosomal RNA.

Expansion segment ES6 in 18S ribosomal RNA is, unlike many other expansion segments, present in all eukaryotes. The available data suggest that ES6 is located on the surface of the small ribosomal subunit. Here we have analyzed the secondary structure of the complete ES6 sequence in intact ribosomes from three eukaryotes, wheat, yeast, and mouse, representing different eukaryotic kingdoms. The availability of the ES6 sequence for modification and cleavage by structure sensitive chemicals and enzymatic reagents was analyzed by primer extension and gel electrophoresis on an ABI 377 automated DNA sequencer. The experimental results were used to restrict the number of possible secondary structure models of ES6 generated by the folding software MFOLD. The modification data obtained from the three experimental organisms were very similar despite the sequence variation. Consequently, similar secondary structure models were obtained for the ES6 sequence in wheat, yeast, and mouse ribosomes. A comparison of sequence data from more than 6000 eukaryotes showed that similar structural elements could also be formed in other organisms. The comparative analysis also showed that the extent of compensatory base changes in the suggested helices was low. The in situ structure analysis was complemented by a secondary structure analysis of wheat ES6 transcribed and folded in vitro. The obtained modification data indicate that the secondary structure of the in vitro transcribed sequence differs from that observed in the intact ribosome. These results suggest that chaperones, ribosomal proteins, and/or tertiary rRNA interactions could be involved in the in vivo folding of ES6.

Animals↗

Complete genome sequence of an aerobic thermoacidophilic crenarchaeon, Sulfolobus tokodaii strain7.

The complete genomic sequence of an aerobic thermoacidophilic crenarchaeon, Sulfolobus tokodaii strain7 which optimally grows at 80 degrees C, at low pH, and under aerobic conditions, has been determined by the whole genome shotgun method with slight modifications. The genomic size was 2,694,756 bp long and the G + C content was 32.8%. The following RNA-coding genes were identified: a single 16S-23S rRNA cluster, one 5S rRNA gene and 46 tRNA genes (including 24 intron-containing tRNA genes). The repetitive sequences identified were SR-type repetitive sequences, long dispersed-type repetitive sequences and Tn-like repetitive elements. The genome contained 2826 potential protein-coding regions (open reading frames, ORFs). By similarity search against public databases, 911 (32.2%) ORFs were related to functional assigned genes, 921 (32.6%) were related to conserved ORFs of unknown function, 145 (5.1%) contained some motifs, and remaining 849 (30.0%) did not show any significant similarity to the registered sequences. The ORFs with functional assignments included the candidate genes involved in sulfide metabolism, the TCA cycle and the respiratory chain. Sequence comparison provided evidence suggesting the integration of plasmid, rearrangement of genomic structure, and duplication of genomic regions that may be responsible for the larger genomic size of the S. tokodaii strain7 genome. The genome contained eukaryote-type genes which were not identified in other archaea and lacked the CCA sequence in the tRNA genes. The result suggests that this strain is closer to eukaryotes among the archaea strains so far sequenced. The data presented in this paper are also available on the internet homepage (http://www.bio.nite.go.jp/E-home/genome_list-e.html/).

Archaeal Proteins↗

Identification and characterization of FTSJ2, a novel human nucleolar protein homologous to bacterial ribosomal RNA methyltransferase.

Cellular RNAs in eukaryotes undergo extensive posttranscriptional modifications, but as yet only a few RNA-modifying enzymes have been identified and characterized. Here we report on the cloning of FTSJ2, a novel human gene encoding a putative RNA methyltransferase. FTSJ2 shares significant sequence homology with FtsJ/RrmJ, a recently identified Escherichia coli 23S rRNA uridine-2'-O-methyltransferase. FTSJ2 belongs to a new family of evolutionarily conserved S-adenosylmethionine-binding proteins. The gene FTSJ2 is located on chromosome 7p22 between MAD1L1 and NUDT1. It is 8 kb in length, spanning three exons. Northern blot analysis revealed that the FTSJ2 transcripts are abundant in skeletal muscle, placenta, and heart, as well as in cancer cells. Immunofluorescence staining demonstrated that FTSJ2 protein localizes to the nucleolus. Our results suggest that FTSJ2 is likely a nucleolar RNA methyltransferase involved in eukaryotic RNA processing and modification.

Amino Acid Sequence↗

Unusually expanded SSU ribosomal DNA of primary osmotrophic euglenids: molecular evolution and phylogenetic inference.

Expansion segments within eukaryotic nuclear SSU ribosomal RNA have been characterized in many diverse organisms. So far, only a few studies have examined the evolutionary history of SSU rDNA variable regions for monophyletic groups. A euglenozoan SSU rDNA data set was analyzed combining phylogenetic inference and examination of expansion segment evolution. Although SSU rDNA length expansion could be ascribed to all Euglenozoa, most unusual length variation occurs within primary osmotrophic euglenids, particularly within the genus Distigma. The longest SSU rRNA gene reported to date can be found in D. sennii, comprising more than 4500 bases. RT-PCR analyses revealed that the complete gene is transcribed into RNA without posttranscriptional modifications. Further investigations uncovered that most of the length extension is due to elongated variable regions V2 and V4, but virtually all variable regions except for V3 are extended within primary osmotrophic euglenids. Analyses of secondary structure revealed several insertion points within variable regions, some of which are of phylogenetic importance. Varying GC content has been detected among species and between expansion segments and core regions. Nevertheless, individual expansion segments of one species as well as variable sequence positions within core regions tend to evolve in parallel concerning nucleotide frequencies. The presence of a large internal repeat within V2 of Distigma sennii hints at a possible mechanism for large-scale sequence length expansion.

Animals↗

Conformation of methylated sequences in HeLa cell 18-S ribosomal RNA: nuclease S1 as a probe.

18-S rRNA from HeLa cells was digested with nuclease S1. Under the conditions employed 15% of the total nucleotides and some 50% of the methylated nucleotides were released as low-molecular-weight products. The material which was precipitable by 70% ethanol after nuclease S1 digestion was subjected to further digestion by combined T1 plus pancreatic ribonucleases or by T1 ribonuclease alone, and fingerprints were prepared. It was found that the four sites which are modified late during ribosome maturation, and which contain base modifications, were all accessible to nuclease S1. By contrast fewer than one-half of the sites which are modified early during ribosome maturation, and which contain 2'-O-methyl groups, were accessible to nuclease S1; the remainder were protected, presumably by secondary or tertiary interactions within 18-S rRNA.

Base Sequence↗

Effects of Histone Deacetylase Inhibitor (HDACi); Trichostatin-A (TSA) on the expression of housekeeping genes.

In quantitative RT-PCR (qRT-PCR), analysis of gene expression is dependent on normalization using housekeeping genes such as 18S rRNA, GAPDH and beta actin. However, variability in their expression has been reported to be caused by factors like drug treatment, pathological states and cell-cycle phase. An emerging area of cancer research focuses on identifying the role of epigenetic alterations such as histone modifications and DNA methylation in the initiation and progression of cancer. Histone acetylation is the best studied modification so far and has been probed through the use of histone deacetylase inhibitors (HDACi). Further, modulation of histone acetylation is currently being explored as a therapeutic strategy in the treatment of cancer and HDACis have shown promise in inhibiting tumorigenesis and metastasis. Trichostatin-A (TSA) is the most widely used HDACi. Therefore, we were driven to identify a suitable internal control for RT-PCR following TSA treatment. We performed quantitative RT-PCR analysis using mouse prostate tissue explants, human prostate cancer (LNCaP) cells and human breast cancer (T-47D and ZR-75-1) cells following TSA treatment. Expression of housekeeping genes including 18S rRNA, beta actin, GAPDH and ribosomal highly-basic 23-kDa protein (rb 23-kDa, RPL13A) were compared in vehicle versus TSA treated samples. Our results showed marked variations in 18S rRNA, beta actin mRNA and GAPDH mRNA levels in mouse prostate explants and a human prostate cancer (LNCaP) cell line following TSA treatment. Furthermore, in two human breast cancer cell lines (T-47D and ZR-75-1) 18S rRNA, beta actin mRNA and GAPDH mRNA levels varied significantly. However, RPL13A mRNA levels remained constant in all the conditions tested. Therefore, we recommend use of RPL13A as a standard for normalization during TSA treatment.

Actins↗

ModiCal: A Targeted Calibration Workflow for Site-Specific m5C Validation by Nanopore Direct RNA Sequencing.

Accurate identification of RNA 5-methylcytidine (m5C) at the single-nucleotide resolution remains a central challenge in nanopore direct RNA sequencing (DRS). Current global scanning and modification-aware basecalling methods enable transcriptome-wide profiling but often yield high false-positive rates and lack site-specific accuracy. To address this, we repurposed ModiDeC, originally a de novo multimodification classifier, into a targeted, high-precision validation tool for RNA modification sites with prior biochemical knowledge. This was implemented through a three-step calibration workflow that alternates between biochemical and computational modules using the well-characterized m5C2278 site in 25S rRNA as a starting point. Baseline training uses short synthetic RNAs carrying either a methylated or unmodified C2278 as ground truth, followed by IVT-derived calibration and validation in methyltransferase knockout yeast. The baseline model accurately detected the bona fide m5C2278 site but initially produced off-target predictions. Iterative retraining with unmodified IVT signals progressively reduced and ultimately eliminated false positives while maintaining a strong signal at the bona fide site. The final model retained enzyme-dependent detection in wild-type versus knockout yeast and, when explicitly targeted, was also able to detect the second rRNA site, C2870, which remained invisible in the initial analysis. Application to native human prerRNA processing intermediates further resolved two distinct m5C deposition regimes on 28S rRNA, while generalization to dengue virus genomic RNA confirmed that the same calibration logic transfers across diverse RNA contexts. Together, this study establishes a reproducible and transferable framework that integrates biochemical validation with iterative neural network refinement, providing a route toward reliable site-specific m5C confirmation by nanopore direct RNA sequencing.

RNA Methylation↗

The accessibility of yeast ribosomal protein L1 as probed by proteolysis and site-directed mutagenesis is different in intact 60 and 80 S ribosome.

Accessible regions of protein L1 in intact 60 and 80 S ribosomes from Saccharomyces cerevisiae were first detected by controlled proteolysis. The N-terminal region of L1 in either 60 S or 80 S particles, was inaccessible to proteases, but the central and C-terminal regions were accessible. The accessibility of the central region differed depending on the ribosome state. These regions were further examined by determination of the chemical reactivity of specific cysteine residues introduced into these regions by site-directed mutagenesis. All cysteine mutant proteins were capable of binding yeast 5 S rRNA in vitro and the ribosomes containing the mutant proteins were functional in vivo. Residues Cys-257 and Cys-275 were modified in both the 60 and 80 S ribosomes but the modification rates were different in the two ribosome states. Both residues Cys-62 and Cys-286 were inaccessible in 80 S or 60 S ribosomes. Taken together, the present study identified several accessible regions of L1 in intact ribosomes and further showed that the accessibility of some of the regions was altered upon ribosomal subunit association. The most likely interpretation of these results is that the conformation of the ribosomal protein L1 was altered upon ribosomal subunit association.

Amino Acid Sequence↗

Chemical modification of cytosine residues of mouse 5 S ribosomal RNA with hydrogen sulfide. (Nucleosides and nucleotides 43).

Cytosine residues of nucleic acids were converted to 4-thiouracil residues with hydrogen sulfide in pyridine and water to examine the secondary and tertiary structures of mouse 5 S rRNA. The cytosine residues at positions 10, 24, 34 (or 36), 39, 44 (or 46) and 63 were converted preferentially when the treatment was carried out at 28 degrees C. This result supports the model of the secondary structure of 5 S rRNA of Nishikawa, K. and Takemura, S. ((1974) FEBS Lett. 40, 106-109) consisting of five helices and five loops. As the temperature was increased to 35 degrees C, additional cytosine residues in positions 26, 52 and 78 were modified to moderate extents.

Animals↗