Dietary cellulose, fructooligosaccharides, and pectin modify fecal protein catabolites and microbial populations in adult cats.

Twelve young adult (1.7 +/- 0.1 yr) male cats were used in a replicated 3 x 3 Latin square design to determine the effects of fiber type on nutrient digestibility, fermentative end products, and fecal microbial populations. Three diets containing 4% cellulose, fructooligosaccharides (FOS), or pectin were evaluated. Feces were scored based on the 5-point system: 1 being hard, dry pellets, and 5 being watery liquid that can be poured. No differences were observed (P > 0.100) in intake of DM, OM, CP, or acid-hydrolyzed fat; DM or OM digestibility; or fecal pH, DM%, output on an as-is or DM basis, or concentrations of histamine or phenylalanine. Crude protein and fat digestibility decreased (P = 0.079 and 0.001, respectively) in response to supplementation with pectin compared with cellulose. Both FOS and pectin supplementation resulted in increased fecal scores (P < 0.001) and concentrations of ammonia (P = 0.003) and 4-methyl phenol (P = 0.003). Fecal indole concentrations increased (P = 0.049) when cats were supplemented with FOS. Fecal acetate (P = 0.030), propionate (P = 0.035), and total short-chain fatty acid (P = 0.016) concentrations increased in pectin-supplemented cats. Fecal butyrate (P = 0.010), isobutyrate (P = 0.011), isovalerate (P = 0.012), valerate (P = 0.026), and total branched-chain fatty acids + valerate (P = 0.008) concentrations increased with supplementation of FOS and pectin. Fecal cadaverine (P < 0.001) and tryptamine (P < 0.001) concentrations increased with supplementation of FOS and pectin. Fecal tyramine concentrations decreased (P = 0.039) in FOS-supplemented cats, whereas spermidine concentrations increased (P < 0.001) in pectin-supplemented cats. Whereas fecal concentrations of putrescine (P < 0.001) and total biogenic amines (P < 0.001) increased with FOS and pectin, the concentrations of these compounds were increased (P < 0.001) in cats supplemented with pectin. Fecal Bifidobacterium spp. concentrations increased (P = 0.006) and Escherichia coli concentrations decreased (P < 0.001) in FOS-supplemented cats. Fecal concentrations of Clostridium perfringens (P < 0.001), E. coli (P < 0.001), and Lactobacillus spp. (P = 0.030) also increased in pectin-supplemented cats. In addition to increasing populations of protein-fermenting microbiota, pectin increased production of fermentative end products associated with carbohydrate compared with protein fermentation. Pectin and FOS may be useful fiber sources in promoting intestinal health of the cat.

Dietary protein concentration affects intestinal microbiota of adult cats: a study using DGGE and qPCR to evaluate differences in microbial populations in the feline gastrointestinal tract.

The objective of this study was to identify qualitative and quantitative differences in microbial populations of adult cats fed diets containing different protein concentrations. Following a 4 week baseline period, eight healthy adult domestic short-hair queens (>1-year-old) were randomly allotted to a moderate-protein (MP; n = 4) or high-protein (HP; n = 4) diet for 8 weeks. Fresh faecal samples were collected after baseline and 8 weeks on treatment and stored at -80 degrees C. Following DNA extraction, samples were analyzed using denaturing gradient gel electrophoresis to distinguish qualitative changes between diets. Quantitative polymerase chain reaction was used to measure E. coli, Bifidobacterium, Clostridium perfringens, and Lactobacillus populations. Compared to baseline, cats fed MP had a bacterial similarity index of 66.7% as opposed to 40.6% similarity for those fed HP, exhibiting marked changes in intestinal bacteria of cats fed HP. Bifidobacterium populations were greater (p < 0.05) in cats fed MP versus HP (9.44 vs. 5.63 CFU/g). Clostridium perfringens populations were greater (p < 0.05) in cats fed HP than MP (12.39 vs. 10.83 CFU/g). In this experiment, a high-protein diet resulted in a dramatic shift in microbial populations. Decreased Bifidobacterium population in cats fed HP may justify prebiotic supplementation for such diets.

Impact of ovariohysterectomy and food intake on body composition, physical activity, and adipose gene expression in cats.

The mechanisms contributing to BW gain following ovariohysterectomy in domestic cats are poorly understood. Moreover, the effects of food restriction to maintain BW following spaying have been poorly studied. Thus, our primary objective was to determine the effects of spaying and food restriction to maintain BW on adipose and skeletal muscle mRNA abundance and activity levels in cats. After a 4-wk baseline period (wk 0), 8 adult (approximately 1.5 yr old) domestic shorthair cats were spayed and fed to maintain BW for 12 wk. After 12 wk, cats were fed ad libitum for an additional 12 wk. Body composition was determined, activity levels were measured, and adipose and muscle biopsies were collected at wk 0, 12, and 24. Fasting blood samples were collected at wk 0, 6, 12, 18, and 24. To maintain BW post-spay, food intake was decreased (P < 0.05) by 30%. During this phase, mRNA abundance of adipose tissue lipoprotein lipase and leptin was decreased (P < 0.05), representing only 52 and 23% of baseline expression, respectively. Interleukin-6 mRNA, however, was increased (P < 0.05) 2-fold. Physical activity was decreased (P < 0.05) by wk 12, most dramatically during the dark period (approximately 20% of baseline activity). During ad libitum feeding (wk 12 to 24), food intake, BW, body fat percentage, and total fat mass were greatly increased (P < 0.05). Compared with wk 0, circulating leptin concentrations tended to increase (P < 0.10) by wk 18 and 24 (4.45 vs. 10.02 and 9.14 ng/mL, respectively), whereas glucose (91 vs. 162 mg/dL) and triacylglyceride (30 vs. 48 mg/dL) concentrations were increased (P < 0.05) by wk 24. Adipose tissue lipoprotein lipase, hormone sensitive lipase, and adiponectin mRNA were decreased (P < 0.05) at wk 24. Adipose interleukin-6 mRNA was increased (P < 0.05) at 24 wk. Physical activity was further decreased (P < 0.05) by wk 24, during the light (60% of baseline) and dark (33% of baseline) periods. In summary, spaying and food restriction affect physical activity levels and several genes associated with lipid metabolism (decreased lipoprotein lipase), food intake (decreased leptin expression), and insulin insensitivity (increased interleukin-6). By identifying these changes, targets for nutritional intervention or lifestyle management have been identified that may curb the risk of obesity and related disorders in spayed cats.

Ovariohysterectomy alters body composition and adipose and skeletal muscle gene expression in cats fed a high-protein or moderate-protein diet.

The objective of this study was to measure changes in body composition, physical activity and adipose and skeletal muscle gene expression of cats fed a high-protein (HP) diet or moderate-protein (MP) diet, following ovariohysterectomy. Eight cats were randomized onto HP or MP diets and were fed those diets for several months prior to baseline. All cats underwent an ovariohysterectomy at baseline (week 0) and were allowed ad libitum access to dietary treatments for 24 weeks. Food intake was measured daily, and BW and body condition score were measured weekly. Blood, adipose and skeletal muscle tissue samples were collected, physical activity was measured, and body composition was determined using DEXA (dual-energy X-ray absorptiometry) at weeks 0, 12 and 24. Caloric intake increased soon after ovariohysterectomy, resulting in increased (P < 0.05) BW at weeks 12 and 24 compared to week 0. Body condition score and body fat percentage increased (P < 0.05) over time. Blood glucose increased (P < 0.05) linearly over time. Non-esterified fatty acids were decreased (P < 0.05) at weeks 12 and 24 compared to week 0. Blood leptin increased (P < 0.05) over time. Total physical activity decreased (P < 0.05) from week 0 to weeks 12 and 24 in all cats. Adipose tissue mRNA abundance of adiponectin, hormone sensitive lipase, toll-like receptor-4, uncoupling protein-2 (UCP2) and vascular endothelial growth factor decreased (P < 0.05) linearly over time, regardless of diet. Skeletal muscle mRNA abundance for glucose transporter-1, hormone sensitive lipase and UCP2 were decreased (P < 0.05), regardless of dietary treatment. Our research noted metabolic changes following ovariohysterectomy that are in agreement with gene expression changes pertaining to lipid metabolism. Feeding cats ad libitum after ovariohysterectomy is inadvisable.

Locked nucleic acid: high-affinity targeting of complementary RNA for RNomics.

Locked nucleic acid (LNA) is a nucleic acid analog containing one or more LNA nucleotide monomers with a bicyclic furanose unit locked in an RNA-mimicking sugar conformation. This conformational restriction is translated into unprecedented hybridization affinity towards complementary single-stranded RNA molecules. That makes fully modified LNAs, LNA/DNA mixmers, or LNA/RNA mixmers uniquely suited for mimicking RNA structures and for RNA targeting in vitro or in vivo. The focus of this chapter is on LNA antisense, LNA-modified DNAzymes (LNAzymes), LNA-modified small interfering (si)RNA (siLNA), LNA-enhanced expression profiling by real-time RT-PCR and detection and analysis of microRNAs by LNA-modified probes.

Improved RNA cleavage by LNAzyme derivatives of DNAzymes.

Specific cleavage of RNA is catalysed by short oligodeoxynucleotides termed DNAzymes. DNAzymes consist of two binding arms that hybridize to a predetermined RNA sequence and a catalytic core that cleaves a phosphodiester bond held between the binding arms. DNAzymes are exemplified by the well-studied 10-23 DNAzyme, which compared with protein ribonucleases is highly specific, albeit slow. Here we report a significant improvement in cleavage kinetics, while maintaining specificity, by incorporation of LNA (locked nucleic acid) and alpha-L-LNA nucleotides into the binding arms of 10-23 DNAzyme. DNAzymes modified in this way (LNAzymes) enhance cleavage of a phosphodiester bond presented in a short RNA substrate as well as in longer and highly structured substrates, and efficient cleavage is maintained from single- to multiple-turnover conditions. Analysis of the cleavage reaction indicates that substrate hybridization is boosted by the presence of the locked residues within the LNAzymes, while no apparent change occurs in the catalytic strand-scission step.

Posttranscriptional modifications in the A-loop of 23S rRNAs from selected archaea and eubacteria.

Posttranscriptional modifications were mapped in helices 90-92 of 23S rRNA from the following phylogenetically diverse organisms: Haloarcula marismortui, Sulfolobus acidocaldarius, Bacillus subtilis, and Bacillus stearothermophilus. Helix 92 is a component of the ribosomal A-site, which contacts the aminoacyl-tRNA during protein synthesis, implying that posttranscriptional modifications in helices 90-92 may be important for ribosome function. RNA fragments were isolated from 23S rRNA by site-directed RNase H digestion. A novel method of mapping modifications by analysis of short, nucleotide-specific, RNase digestion fragments with Matrix Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS) was utilized. The MALDI-MS data were complemented by two primer extension techniques using reverse transcriptase. One technique utilizes decreasing concentrations of deoxynucleotide triphosphates to map 2'-O-ribose methylations. In the other, the rRNA is chemically modified, followed by mild alkaline hydrolysis to map pseudouridines (psis). A total of 10 posttranscriptionally methylated nucleotides and 6 psis were detected in the five organisms. Eight of the methylated nucleotides and one psi have not been reported previously. The distribution of modified nucleotides and their locations on the surface of the ribosomal peptidyl transferase cleft suggests functional importance.

The pleuromutilin drugs tiamulin and valnemulin bind to the RNA at the peptidyl transferase centre on the ribosome.

The pleuromutilin antibiotic derivatives, tiamulin and valnemulin, inhibit protein synthesis by binding to the 50S ribosomal subunit of bacteria. The action and binding site of tiamulin and valnemulin was further characterized on Escherichia coli ribosomes. It was revealed that these drugs are strong inhibitors of peptidyl transferase and interact with domain V of 23S RNA, giving clear chemical footprints at nucleotides A2058-9, U2506 and U2584-5. Most of these nucleotides are highly conserved phylogenetically and functionally important, and all of them are at or near the peptidyl transferase centre and have been associated with binding of several antibiotics. Competitive footprinting shows that tiamulin and valnemulin can bind concurrently with the macrolide erythromycin but compete with the macrolide carbomycin, which is a peptidyl transferase inhibitor. We infer from these and previous results that tiamulin and valnemulin interact with the rRNA in the peptidyl transferase slot on the ribosomes in which they prevent the correct positioning of the CCA-ends of tRNAs for peptide transfer.

Inhibition of the ribosomal peptidyl transferase reaction by the mycarose moiety of the antibiotics carbomycin, spiramycin and tylosin.

Many antibiotics, including the macrolides, inhibit protein synthesis by binding to ribosomes. Only some of the macrolides affect the peptidyl transferase reaction. The 16-member ring macrolide antibiotics carbomycin, spiramycin, and tylosin inhibit peptidyl transferase. All these have a disaccharide at position 5 in the lactone ring with a mycarose moiety. We have investigated the functional role of this mycarose moiety. The 14-member ring macrolide erythromycin and the 16-member ring macrolides desmycosin and chalcomycin do not inhibit the peptidyl transferase reaction. These drugs have a monosaccharide at position 5 in the lactone ring. The presence of mycarose was correlated with inhibition of peptidyl transferase, footprints on 23 S rRNA and whether the macrolide can compete with binding of hygromycin A to the ribosome. The binding sites of the macrolides to Escherichia coli ribosomes were investigated by chemical probing of domains II and V of 23 S rRNA. The common binding site is around position A2058, while effects on U2506 depend on the presence of the mycarose sugar. Also, protection at position A752 indicates that a mycinose moiety at position 14 in 16-member ring macrolides interact with hairpin 35 in domain II. Competitive footprinting of ribosomal binding of hygromycin A and macrolides showed that tylosin and spiramycin reduce the hygromycin A protections of nucleotides in 23 S rRNA and that carbomycin abolishes its binding. In contrast, the macrolides that do not inhibit the peptidyl transferase reaction bind to the ribosomes concurrently with hygromycin A. Data are presented to argue that a disaccharide at position 5 in the lactone ring of macrolides is essential for inhibition of peptide bond formation and that the mycarose moiety is placed near the conserved U2506 in the central loop region of domain V 23 S rRNA.

Negative in vitro selection identifies the rRNA recognition motif for ErmE methyltransferase.

Erm methyltransferases modify bacterial 23S ribosomal RNA at adenosine 2058 (A2058, Escherichia coli numbering) conferring resistance to macrolide, lincosamide, and streptogramin B (MLS) antibiotics. The motif that is recognized by Erm methyltransferases is contained within helix 73 of 23S rRNA and the adjacent single-stranded region around A2058. An RNA transcript of 72 nt that displays this motif functions as an efficient substrate for the ErmE methyltransferase. Pools of degenerate RNAs were formed by doping 34-nt positions that extend over and beyond the putative Erm recognition motif within the 72-mer RNA. The RNAs were passed through a series of rounds of methylation with ErmE. After each round, RNAs were selected that had partially or completely lost their ability to be methylated. After several rounds of methylation/selection, 187 subclones were analyzed. Forty-three of the subclones contained substitutions at single sites, and these are confined to 12 nucleotide positions. These nucleotides, corresponding to A2051-A2060, C2611, and A2614 in 23S rRNA, presumably comprise the RNA recognition motif for ErmE methyltransferase. The structure formed by these nucleotides is highly conserved throughout bacterial rRNAs, and is proposed to constitute the motif that is recognized by all the Erm methyltransferases.

Early gene expression of NK cell-activating chemokines in mice resistant to Leishmania major.

Susceptibility of mice to Leishmania major is associated with an insufficient NK cell-mediated innate immune response. We analyzed the expression of NK cell-activating chemokines in vivo during the first days of infection in resistant and susceptible mice. The mRNA expression of gamma interferon-inducible protein 10 (IP-10), monocyte chemoattractant protein 1 (MCP-1), and lymphotactin was upregulated 1 day after infection in the draining lymph nodes of resistant C57BL/6 mice but not in those of susceptible BALB/c mice. In vivo local treatment of BALB/c mice with recombinant IP-10 shortly after infection resulted in an enhanced NK cell activity in the draining lymph node. The data suggest that although the recruitment of NK cells is normal in susceptible mice, the lack of NK cell-activating chemokines is a factor resulting in a suboptimal NK cell-mediated defense.

ErmE methyltransferase recognizes features of the primary and secondary structure in a motif within domain V of 23 S rRNA.

The Erm methyltransferases confer resistance to macrolide, lincosamide and streptogramin B (MLS) antibiotics by methylation of a single adenosine base within bacterial 23 S ribosomal RNA. The ErmE methyltransferase, from the macrolide-producing bacterium Saccharopolyspora erythraea, recognizes a motif within domain V of the rRNA that specifically targets adenosine 2058 (A2058) for methylation. Here, we define the structure of the RNA motif by a combination of molecular genetics and biochemical probing. The core of the motif has the primary sequence 2056-GGAHA-2060, where H is any nucleotide except guanosine, and ErmE methylates at the adenosine in bold. For efficient recognition by ErmE, this sequence must be displayed within a particular secondary structure. An irregular stem (helix 73) is required immediately 5' to A2058, with an unpaired nucleotide, preferably a cytidine residue, at position 2055. Nucleotides 2611 to 2616 are collectively required to form part of the 3'-side of helix 73, but there is little or no restriction on the identities of individual nucleotides here. There are minor preferences in the identities of nucleotides 2051 to 2055 that are adjacent to the motif core, although their main role is in maintaining the irregular secondary structure. The essential elements of the ErmE motif are conserved in bacterial 23 S rRNAs, and thus presumably also form the recognition motif for other Erm methyltransferases.

Core sequence in the RNA motif recognized by the ErmE methyltransferase revealed by relaxing the fidelity of the enzyme for its target.

Under physiological conditions, the ErmE methyltransferase specifically modifies a single adenosine within ribosomal RNA (rRNA), and thereby confers resistance to multiple antibiotics. The adenosine (A2058 in Escherichia coli 23S rRNA) lies within a highly conserved structure, and is methylated efficiently, and with equally high fidelity, in rRNAs from phylogenetically diverse bacteria. However, the fidelity of ErmE is reduced when magnesium is removed, and over twenty new sites of ErmE methylation appear in E. coli 16S and 23S rRNAs. These sites show widely different degrees of reactivity to ErmE. The canonical A2058 site is largely unaffected by magnesium depletion and remains the most reactive site in the rRNA. This suggests that methylation at the new sites results from changes in the RNA substrate rather than the methyltransferase. Chemical probing confirms that the rRNA structure opens upon magnesium depletion, exposing potential new interaction sites to the enzyme. The new ErmE sites show homology with the canonical A2058 site, and have the consensus sequence aNNNcgGAHAg (ErmE methylation occurs exclusively at adenosines (underlined); these are preceded by a guanosine, equivalent to G2057; there is a high preference for the adenosine equivalent to A2060; H is any nucleotide except G; N is any nucleotide; and there are slight preferences for the nucleotides shown in lower case). This consensus is believed to represent the core of the motif that Erm methyltransferases recognize at their canonical A2058 site. The data also reveal constraints on the higher order structure of the motif that affect methyltransferase recognition.

ErmE methyltransferase recognition elements in RNA substrates.

Dimethylation by Erm methyltransferases at the N-6 position of adenine 2058 (A2058, Escherichia coli numbering) in domain V of bacterial 23 S rRNA confers resistance to the macrolide-lincosamide-streptogramin B (MLS) group of antibiotics. The ErmE methyltransferase from Saccharopolyspora erythraea methylates a 625 nucleotide transcript of domain V as efficiently as it methylates intact 23 S rRNA. By progressively truncating domain V, the motif required for specific recognition by the enzyme has been localized to a helix and single-stranded region adjacent to A2058. The smallest RNA transcript that shows methyl-accepting activity is a 27-nucleotide stem-loop, corresponding to the 23 S rRNA sequences 2048 to 2063 and 2610 to 2620 (helix 73), with A2058 situated within the hairpin loop. Methylation of A2058 in the truncated RNAs is optimal in the absence of magnesium, and the efficiency of methylation is halved by the presence of 2 to 3 mM magnesium. Magnesium serves to stabilize a conformation in the truncated RNA that prevents efficient methylation. This contrasts to the intact domain V RNA, where 2 mM magnesium ions support a conformation at A2058 that is most readily recognized by ErmE. Methylation of domain V RNA is generally far less susceptible to ionic conditions than the truncated RNAs. The effects of monovalent cations on the methylation of truncated transcripts suggest that RNA structures outside helix 73 support the ErmE interaction. However, interaction with these structures is not essential for specific ErmE recognition of A2058.

Movement of the 3'-end of tRNA through the peptidyl transferase centre and its inhibition by antibiotics.

Determining how antibiotics inhibit ribosomal activity requires a detailed understanding of the interactions and relative movement of tRNA, mRNA and the ribosome. Recent models for the formation of hybrid tRNA binding sites during the elongation cycle have provided a basis for re-evaluating earlier experimental data and, especially, those relevant to substrate movements through the peptidyl transferase centre. With the exception of deacylated tRNA, which binds at the E-site, ribosomal interactions of the 3'-ends of the tRNA substrates generate only a small part of the total free energy of tRNA-ribosome binding. Nevertheless, these relatively weak interactions determine the unidirectional movement of tRNAs through the ribosome and, moreover, they appear to be particularly susceptible to perturbation by antibiotics. Here we summarise current ideas relating particularly to the movement of the 3'-ends of tRNA through the ribosome and consider possible inhibitory mechanisms of the peptidyl transferase antibiotics.

Recognition determinants for proteins and antibiotics within 23S rRNA.

Ribosomal RNAs fold into phylogenetically conserved secondary and tertiary structures that determine their function in protein synthesis. We have investigated Escherichia coli 23S rRNA to identify structural elements that interact with antibiotic and protein ligands. Using a combination of molecular genetic and biochemical probing techniques, we have concentrated on regions of the rRNA that are connected with specific functions. These are located in different domains within the 23S rRNA and include the ribosomal GTPase-associated center in domain II, which contains the binding sites for r-proteins L10.(L12)4 and L11 and is inhibited by interaction with the antibiotic thiostrepton. The peptidyltransferase center within domain V is inhibited by macrolide, lincosamide, and streptogramin B antibiotics, which interact with the rRNA around nucleotide A2058. Drug resistance is conferred by mutations here and by modification of A2058 by ErmE methyltransferase. ErmE recognizes a conserved motif displayed in the primary and secondary structure of the peptidyl transferase loop. Within domain VI of rRNA, the alpha-sarcin stem-loop is associated with elongation factor binding and is the target site for ribotoxins including the N-glycosidase ribosome-inactivating proteins ricin and pokeweed antiviral protein (PAP). The orientations of the 23S rRNA domains are constrained by tetiary interactions, including a pseudoknot in domain II and long-range base pairings in the center of the molecule that bring domains II and V closer together. The phenotypic effects of mutations in these regions have been investigated by expressing 23S rRNA from plasmids. Allele-specific priming sites have been introduced close to these structures in the rRNA to enable us to study the molecular events there.

The conformation of 23S rRNA nucleotide A2058 determines its recognition by the ErmE methyltransferase.

The ErmE methyltransferase confers resistance to MLS antibiotics by specifically dimethylating adenine 2058 (A2058, Escherichia coli numbering) in bacterial 23S rRNA. To define nucleotides in the rRNA that are part of the motif recognized by ErmE, we investigated both in vivo and in vitro the effects of mutations around position A2058 on methylation. Mutagenizing A2058 (to G or U) completely abolishes methylation of 23S rRNA by ErmE. No methylation occurred at other sites in the rRNA, demonstrating the fidelity of ErmE for A2058. Breaking the neighboring G2057-C2611 Watson-Crick base pair by introducing either an A2057 or a U2611 mutation, greatly reduces the rate of methylation at A2058. Methylation remains impaired after these mutations have been combined to create a new A2057-U2611 Watson-Crick base interaction. The conformation of this region in 23S rRNA was probed with chemical reagents and it was shown that the A2057 and U2611 mutations alone and in combination alter the reactivity of A2058 and adjacent bases. However, mutagenizing position G-->A2032 in an adjacent loop, which has been implicated to interact with A2058, alters neither the ErmE methylation at A2058 nor the accessibility of this region to the chemical reagents. The data indicate that a less-exposed conformation at A2058 leads to reduction in methylation by ErmE. Nucleotide G2057 and its interaction with C2611 maintain the conformation at A2058, and are thus important in forming the structural motif that is recognized by the ErmE methyltransferase.

Domain V of 23S rRNA contains all the structural elements necessary for recognition by the ErmE methyltransferase.

The ErmE methyltransferase from the erythromycin-producing actinomycete Saccharopolyspora erythraea dimethylates the N-6 position of adenine 2058 in domain V of 23S rRNA. This modification confers resistance to erythromycin and to other macrolide, lincosamide, and streptogramin B antibiotics. We investigated what structural elements in 23S rRNA are required for specific recognition by the ErmE methyltransferase. The ermE gene was cloned into R1 plasmid derivatives, providing a means of inducible expression in Escherichia coli. Expression of the methyltransferase in vivo confers resistance to erythromycin and clindamycin. The degree of resistance corresponds to the level of ermE expression. In turn, ermE expression also correlates with the proportion of 23S rRNA molecules that are dimethylated at adenine 2058. The methyltransferase was isolated in an active, concentrated form from E. coli, and the enzyme efficiently modifies 23S rRNA in vitro. Removal of most of the 23S rRNA structure, so that only domain V (nucleotides 2000 to 2624) remains, does not affect the efficiency of modification by the methyltransferase. In addition, modification still occurs after the rRNA tertiary structure has been disrupted by removal of magnesium ions. We conclude that the main features that are specifically recognized by the ErmE methyltransferase are displayed within the primary and secondary structures of 23S rRNA domain V.

Presence of a nuclear lamina in pachytene spermatocytes of the rat.

The nuclear lamina is a karyoskeletal structure located at the periphery of cell nuclei. The major constituents are the lamins, which belong to the evolutionarily conserved multigene family of intermediate filament proteins. Lamins show a conspicuous cell type-specific expression pattern. The majority of somatic cells of vertebrates express A-type (lamins A and C) as well as B-type (lamins B1 and B2) lamins. Although a lamina structure has been demonstrated to be a ubiquitous component of somatic nuclei its existence in certain meiotic stages during spermatogenesis has been a matter of debate. In this study, we investigated the expression of lamins in rat spermatogenic cells using immunological and protein-chemical methods. We report on the presence of a nuclear lamina structure in rat pachytene spermatocytes. With the aid of a novel broad-reacting lamin antibody we have demonstrated the expression of a protein that is closely related, if not identical, to lamin B1.