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Molecular subtyping of Borrelia burgdorferi in erythema migrans and acrodermatitis chronica atrophicans.

Recently, three subtypes of Borrelia burgdorferi have been identified: Borrelia burgdorferi sensu stricto, Borrelia garinii, and the VS 461 group of Borrelia burgdorferi. These subtypes differ by nucleotide sequence variations within several Borrelia burgdorferi specific genes and most likely by their pathogenetic potential. To assess whether different subtypes of Borrelia burgdorferi might be associated with different cutaneous manifestations and clinical courses of Lyme disease, lesional skin biopsies from 35 patients with erythema migrans and 18 patients with acrodermatitis chronica atrophicans were analyzed. A Borrelia burgdorferi specific gene segment encoding a 26-kD protein with subtype specific nucleotide sequence variations was amplified by a nested polymerase chain reaction technique. For molecular subtyping, the products were transcribed into complementary RNA. Upon polyacrylamide gel electrophoresis, complementary RNA molecules separate into several metastable conformational forms resulting in patterns of bands highly specific for the nucleotide sequence of the transcribed molecules. In biopsy specimens of erythema migrans, the VS 461 subtype was detected in 28 of 35 and the Borrelia garinii subtype in six of 35 cases. In one of 35 cases of erythema migrans Borrelia burgdorferi sensu stricto as well as Borrelia garinii was detected. In contrast, in all 18 biopsies of acrodermatitis chronica atrophicans, only the VS 461 subtype was identified. This subtype is rarely found in the USA, where acrodermatitis chronica atrophicans is almost unknown. These data indicate that acrodermatitis chronica atrophicans might be closely associated with the VS 461 group of Borrelia burgdorferi.

Acrodermatitis↗

Hybridization of pigeon globin messenger RNA with complementary DNA synthesized in vitro by reverse transcription: influence of the homopolymeric regions.

The kinetics of hybridization of pigeon globin messenger RNA with complementary cDNA synthesized by means of AMV reverse transcriptase is complex. Addition of poly A or poly U in excess to the reaction mixture normalized the kinetics. It is concluded that association of the complementary homopolymeric regions of mRNA and cDNA accelerates the complex formation between heteropolymeric sequences in a fraction of the molecules.

Adenine Nucleotides↗

Toward the therapeutic editing of mutated RNA sequences.

If RNA editing could be rationally directed to mutated RNA sequences, genetic diseases caused by certain base substitutions could be treated. Here we use a synthetic complementary RNA oligonucleotide to direct the correction of a premature stop codon mutation in dystrophin RNA. The complementary RNA oligonucleotide was hybridized to a premature stop codon and the hybrid was treated with nuclear extracts containing the cellular enzyme double-stranded RNA adenosine deaminase. When the treated RNAs were translated in vitro, a dramatic increase in expression of a downstream luciferase coding region was observed. The cDNA sequence data are consistent with deamination of the adenosine in the UAG stop codon to inosine by double-stranded RNA adenosine deaminase. Injection of oligonucleotide-mRNA hybrids into Xenopus embryos also resulted in an increase in luciferase expression. These experiments demonstrate the principle of therapeutic RNA editing.

Adenosine Deaminase↗

Estrogen enhances endometrial estrogen receptor gene expression by a posttranscriptional mechanism in the ovariectomized ewe.

Prior influence of estrogen is required for many physiological effects of steroid hormones. This study addresses positive autoregulation of estrogen receptor gene expression in endometrium. Groups of ovariectomized ewes (n = 6) were treated with a single i.m. injection of 50 micrograms 17 beta-estradiol for 6, 12, 24, or 48 h or of vehicle for 24 h (control) prior to collection of endometrium. Three ewes received a regimen of estradiol-progesterone-estradiol (EPE) designed to mimic the estrous cycle. Northern analysis of endometrial RNA using an ovine estrogen receptor complementary RNA probe indicated that estradiol increased (p < 0.0001) estrogen receptor messenger RNA abundance over time to be 5-fold greater at 24 h postinjection. This effect also occurred after a period of progesterone dominance in EPE ewes (p < 0.05). Ribonuclease protection assays with a complementary RNA probe for the ovine progesterone receptor demonstrated that estradiol treatment increased progesterone receptor messenger RNA abundance at 48 h (p < 0.005). Nuclear runoff analyses indicated that whereas estradiol enhanced the transcription rates of progesterone receptor (p < 0.1) and 28S ribosomal RNA genes (p < 0.002), activity of the estrogen receptor gene was unchanged (p > 0.25). These results suggest that a physiological dose of estradiol, similar to the preovulatory surge of estrogen, up-regulates endometrial estrogen receptor gene expression by a posttranscriptional mechanism.

Amino Acid Sequence↗

Plasmid library for the transcription of RNA probes complementary to the entire genome of the human immunodeficiency virus type 1 (HIV-1).

A plasmid (pBH10R3) containing a 9-kb Sst I fragment of HIV-1 (clone BH-10) inserted in pSP64, an in vitro expression vector, has been used for the transcription of anti-sense HIV-1 RNA. With this system, the transcripts obtained in vitro were not usually full length (1 to 2 kb long) and they predominantly span the 3' end ORF and ENV regions of the viral genome. We have rearranged the HIV-1 genomic sequences with respect to the SP6 promoter in the pSP64 vector and have obtained a series of new constructs allowing the expression in vitro of RNA transcripts complementary to other regions in the HIV-1 genome, including the 5' end of the ENV region as well as the TAT, POL, and GAG regions. In fact, the combined use of these constructs as templates for in vitro transcription allows the production of RNA probes spanning the entire viral genome. Compared with the 1- to 2-kb probes mentioned above, the combined use of such probes results in a several-fold increase in the sensitivity of molecular hybridization for the detection of HIV-1 nucleic acid sequences. Also, these constructs enable the preparation of RNA probes that have the potential to detect restriction polymorphisms throughout the HIV-1 genome.

Gene Library↗

Phosphorothioate oligonucleotides are inhibitors of human DNA polymerases and RNase H: implications for antisense technology.

Phosphorothioate oligodeoxycytidine (S-dCn) was used as a model compound to examine the impact of the number of phosphorothioate linkages and their position on the inhibition of human DNA polymerases and RNase H in vitro. S-dCn with a chain length longer than 15 could inhibit human DNA polymerases and RNase H activities, in a linkage number-dependent manner. Longer oligomers were more potent inhibitors than shorter ones. Kinetic studies indicated that S-dC28 was a competitive inhibitor of DNA polymerase alpha and beta with respect to the DNA template, whereas it was a noncompetitive inhibitor of polymerases gamma and delta. S-dC28 was also a competitive inhibitor of RNase H1 and H2 with respect to RNA-DNA duplex. Susceptibility of these enzymes to inhibition by S-dC28 was in the order of delta approximately gamma greater than alpha greater than beta and RNase H1 greater than RNase H2. Structural-activity relationships were explored with a group of S-dC28 analogs that have phosphorothioate internucleotide linkages at various positions. The inhibitory effect depended on the total number of thioate linkages, rather than the position of the linkages within the oligomer or the chain length itself. No sequence specificity was found. In the presence of the complementary RNA, antisense phosphorothioates (S-oligos) exerted a biphasic effect on RNase H activity. At low concentrations S-oligos could enhance the cleavage of the RNA portion of S-oligo-RNA duplex, whereas at high concentrations (in excess of the complementary RNA) S-oligos could inhibit RNase H and protect the complementary RNA from degradation. Together, these results suggest that the non-sequence-specific inhibitory effect of S-oligos should be taken into consideration in designing antisense inhibitors. This inhibitory activity could be avoided by decreasing the number of phosphorothioate linkages at the backbone, and S-oligos of 15-20 residues are preferable in antisense molecule design.

Base Sequence↗

Synthesis and evaluation of some properties of chimeric oligomers containing PNA and phosphono-PNA residues.

In an attempt to improve physico-chemical and biological properties of peptide nucleic acids (PNAs), particularly water solubility and cellular uptake, the synthesis of chimeric oligomers consisted of PNA and phosphono-PNA analogues (pPNAs) bearing the four natural nucleobases has been accomplished. To produce these chimeras, pPNA monomers of two types containing N-(2-hydroxyethyl)phosphonoglycine, or N-(2-aminoethyl)phosphonoglycine backbone, were used in conjunction with PNA monomers representing derivatives of N-(2-aminoethyl)glycine, or N-(2-hydroxyethyl)glycine. The oligomers obtained were composed of either PNA and pPNA stretches or alternating PNA and pPNA monomers. The examination of hybridization properties of PNA-pPNA chimeras to DNA and RNA complementary strands in comparison with pure PNAs, and pPNAs as well as DNA-pPNA hybrids and DNA fragments confirmed that these chimeras form stable complexes with complementary DNA and RNA fragments. They were found to be resistant to degradation by nucleases. All these properties together with good solubility in water make PNA-pPNA hybrids promising for further evaluation as potential therapeutic agents.

Chemical Phenomena↗

Expression of naturally occurring RNA molecules complementary to the murine L27' ribosomal protein mRNA.

We report here the existence of two naturally occurring RNA molecules that are complementary to the murine L27' ribosomal protein (rp) mRNA. These transcripts are 1.8 and 1.0 kb in length, and are both found in poly(A)+ populations of cytoplasmic and polysomal RNA of a number of established cell lines and in all adult murine tissues examined with the exception of the testes, where only the 1.8-kb transcript was detected. The expression of the 1.8-kb transcript is also constant during mouse embryogenesis from days 11 through 18 of gestation, and during differentiation of P19 embryonal carcinoma cells, whereas that of the smaller transcript decreases at 14 days and was not detected in 16- and 18-day embryos or in differentiated P19 cells. At the structural level both countertranscripts share the same region of perfect or near perfect complementarity to the L27' rp mRNA, which spans more than 75% of the coding region of the latter. The 0.8-kb difference in length of the two countertranscripts lies mainly 3' of the divergence from complementarity to the rp sequence. Indirect evidence suggests that the countertranscripts do not originate from the active L27' rp gene copy. The possible biological significance of the co-existence of the countertranscripts with the housekeeping L27' rp mRNA within the same cell is discussed.

Animals↗

A conserved and unique (AT)-rich segment in yeast mitochondrial DNA.

The mtDNA of the cytoplasmic petite mutant of yeast RD1A consists mainly of a perfect head-to-tail repetition of a known sequence of 66 consecutive AT and 2 GC base pairs. We have hybridized complementary RNA made on RD1A mtDNA with the mtDNAs of four different wild-type Saccharomyces strains that differ markedly in restriction fragmentation pattern. The tm's of the four heteroduplexes are identical to the tm of the homoduplex of RD1A mtDNA with complementary RNA of one repeat length. With all four wild-type mtDNAs this complementary RNA hybridizes mainly to a single restriction fragment of about 300 base pairs. This shows the conservation and individuality of at least one (AT)-rich segment in yeast mtDNA. The 300 base pair fragment has been mapped in the vicinity of the oxi-2 locus. The possible role of the (AT)-rich segment in the processing of the primary transcript of this region is discussed.

Adenine↗

Polyoma virus-specific RNA synthesis in an inducible line of polyoma virus-transformed rat cells.

Viral RNA present in the inducible LPT clone 1A of polyoma virus-transformed rat cells was characterized before and after mitomycin C induction by hybridization with 32P-labeled separated E and L strands of polyoma viral DNA restriction endonuclease fragments. In clone 1A cells maintained under normal growth conditions, the cytoplasm contained a transcript of the E-strand DNA from the "early" region similar to that previously identified in lytically infected cells, as well as minor quantities of RNA complementary to less than one-half of the L- and the E-strand DNA from the "late" region. Nuclei of normally growing cells contained the same species found in the cytoplasm, as well as an additional abundant RNA complementary to one-half of the L-strand DNA of the late region. No significant changes occurred in the cytoplasmic viral RNA after mitomycin C treatment before the onset of viral DNA replication, but the concentration of the nuclear L-strand DNA transcript diminished. After the onset of viral DNA replication after mitomycin C treatment, transcripts of virtually the entire L-strand DNA were found in the nuclei, and a 10-fold increase was observed in the abundance of RNA transcribed from the E strand of the early region. In the cytoplasm, the abundance of the early RNA increased about 25-fold and late RNA complementary to the L-strand DNA of the late region was found in a similar quantity. The synthesis of both the early and the late RNA species was inhibited if viral DNA replication was blocked with 5-fluorodeoxyuridine. We conclude that the induction of viral DNA replication in LPT cells is not determined at the level of mRNA synthesis.

Animals↗

Histone RNA in amphibian oocytes visualized by in situ hybridization to methacrylate-embedded tissue sections.

We present an in situ hybridization method for detecting cellular RNAs in tissue sections using methacrylate as the embedding medium. The technique offers the advantage of superior morphological preservation compared with previously published procedures. Since sections can be cut 1 micron or less in thickness, full advantage is taken of the short path length of 3H electrons. Applying this procedure to developing amphibian oocytes, we investigated the accumulation and localization of RNA complementary to the histone genes and their adjacent spacers. Histone RNA begins to accumulate in the cytoplasm of late pachytene-early diplotene oocytes, rapidly reaching a maximum concentration during Dumont stage 1. After this stage the concentration of histone RNA declines. RNA transcribed from histone coding regions is located almost exclusively in the cytoplasm of oocytes. Transcripts of the spacer regions, which are known to be synthesized on oocyte lampbrush chromosomes, do not accumulate in the oocytes. [3H]RNA complementary to U2 small nuclear RNA, used in these experiments as a control, hybridized predominantly to the nucleus of the oocytes.

Animals↗

Effects of 5-(N-aminohexyl)carbamoyl-2'-deoxyuridine on endonuclease stability and the ability of oligodeoxynucleotide to activate RNase H.

To evaluate an endonuclease resistance property of oligodeoxynucleotides (ODNs) containing 5-(N-aminohexyl)carbamoyl-2'-deoxyuridines (Hs) and to elucidate whether a duplex consisting of the ODN analogue and its complementary RNA induces RNase H activity, the ODNs containing the deoxyuridine analogues, Hs, at intervals of one, two, three, four and five natural nucleosides were synthesized. From partial hydrolysis of these ODNs with nuclease S1 (an endonuclease), it was found that the ODNs became more stable towards nucleolytic hydrolysis by the enzyme as the number of H increased. Furthermore, to examine whether the duplexes composed of the ODNs containing Hs and their complementary RNAs are substrates for RNase H or not, the duplexes of these ODNs and their complementary RNA strands were treated with Escherichia coliRNase H. It was found that cleavage of the RNA strands by the enzyme was kinetically affected by the introduction of Hs into the duplexes.

Base Sequence↗

Watson-Crick base-pairing properties of bicyclo-DNA.

A series of sequences of the DNA analog bicyclo-DNA, 6-12 nucleotides in length and containing all four natural nucleobases, were prepared and their Watson-Crick pairing properties with complementary RNA and DNA, as well as in its own series, were analyzed by UV-melting curves and CD-spectroscopy. The results can be summarized as follows: bicyclo-DNA forms stable Watson-Crick duplexes with complementary RNA and DNA, the duplexes with RNA generally being more stable than those with DNA. Pyrimidine-rich bicyclo-DNA sequences form duplexes of equal or slightly increased stability with DNA or RNA, whereas purine-rich sequences show decreased affinity to complementary DNA and RNA when compared with wild-type (DNA-DNA, DNA-RNA) duplexes. In its own system, bicyclo-DNA prefers antiparallel strand alignment and strongly discriminates for base mismatches. Duplexes are always inferior in stability compared with the natural ones. A detailed analysis of the thermodynamic properties was performed with the sequence 5'-GGATGGGAG-3'x 5'-CTCCCATCC-3' in both backbone systems. Comparison of the pairing enthalpy and entropy terms shows an enthalpic advantage for DNA association (delta deltaH = -18 kcal x (mol)-1)) and an entropic advantage for bicyclo-DNA association (delta deltaS = 49 cal x K(-1) x mol(-1), leading to a delta deltaG 25 degrees C of -3.4 kcal x mol(-1) in favor of the natural duplex. The salt dependence of Tm for this sequence is more pronounced in the case of bicyclo-DNA due to increased counter ion screening from the solvent. Furthermore bicyclo-DNA sequences are more stable towards snake venom phosphodiesterase by a factor of 10-20, and show increased stability in fetal calf serum by a factor of 8 compared with DNA.

Base Composition↗

Hypertrophic scar tissues and fibroblasts produce more transforming growth factor-beta1 mRNA and protein than normal skin and cells.

Transforming growth factor-beta1 is a well-known fibrogenic cytokine produced by many types of cells including dermal fibroblasts. To investigate whether this fibrogenic cytokine is involved in development of hypertrophic scar, transforming growth factor-beta1 gene expression was evaluated in small skin samples. Because a sufficient quantity of normal skin from patients with hypertrophic scar is not readily available, a reverse transcription-polymerase chain reaction technique was used. Quantitation of gene expression by reverse transcription-polymerase chain reaction is difficult partly due to the lack of suitable complementary RNA standards. We have established a convenient, reliable procedure to construct an internal standard for transforming growth factor-beta1 starting with a gene specific polymerase chain reaction product. After digestion of the polymerase chain reaction product with endonuclease, a small piece of cDNA from human procollagen alpha1(I) cDNA with compatible ends was inserted into the polymerase chain reaction-DNA fragment. The recombinant cDNA was re-amplified by polymerase chain reaction and subcloned into a plasmid containing bacteriophage T7 and T3 promoters. Complementary RNA was prepared from the recombinant plasmid and amplified by reverse transcription-polymerase chain reaction together with the tissue or cellular RNA. After amplification, the products were electrophoresed in an agarose gel containing ethidium bromide. The bands for internal standard and transforming growth factor-beta1 mRNA were scanned, digitized, and plotted against the amount of internal standard complementary RNA added in the reverse transcription-polymerase chain reaction. The number of mRNA molecules/cell was calculated. We examined the transforming growth factor-beta1 mRNA in hypertrophic scar tissue and in normal skin and found that hypertrophic scar tissues expressed five-fold more transforming growth factor-beta1 mRNA than normal skin per unit of wet weight. We used this procedure to quantitate transforming growth factor-beta1 mRNA expression in 5 pairs of fibroblast cultures derived from hypertrophic scar and normal skin. The results showed that hypertrophic scar fibroblast cultures contain significantly more molecules of mRNA for transforming growth factor-beta1 than normal cells (116 +/- 6 vs. 97 +/- 7, p = 0.017, n = 5). These results were supported by Northern analysis for transforming growth factor-beta1 mRNA in the cells and enzyme-linked immunosorbent assay for TGF-beta1 protein in fibroblast-conditioned medium. In conclusion, hypertrophic scar tissue and fibroblasts produce more mRNA and protein for transforming growth factor-beta1, which may be important in hypertrophic scar formation. The construction of the gene specific internal standard for reverse transcription-polymerase chain reaction is a simple and reliable procedure useful to quantitate gene expression in a small amount of tissue or number of cells.

Biopsy, Needle↗

Strand orientation of simian virus 40 transcription in productively infected cells.

Strand orientation of transcription in BSC-1 cells infected by simian virus 40 (SV40) was investigated by annealing RNAs extracted from infected cells with asymmetric complementary RNA from SV40 DNA synthesized in vitro by RNA polymerase from Escherichia coli. The results suggest that the early viral RNA sequences (those made before replication of viral DNA) and the late RNA sequences (those synthesized concurrently with replication of viral DNA) are transcribed from opposite strands of the SV40 DNA. The RNA synthesized in vitro is of the same polarity as the early in vivo RNA and is complementary to the late in vivo RNA. Knowledge of strand selection in lytic infection is a step towards understanding the regulation of transcription of this viral DNA.

Animals↗

Growth hormone receptor gene is expressed in neuropeptide Y neurons in hypothalamic arcuate nucleus of rats.

GH feeds back on the hypothalamus and regulates its own secretion. We have previously shown that systemic administration of GH induces expression of the c-fos gene, a marker of neuronal activity, on the hypothalamic neuropeptide Y(NPY) and somatostatin neurons in rats. We argued that if GH were to act directly on NPY neurons, NPY neurons should express the GH receptor (GHR) gene. To test this hypothesis, coronal sections of the medial basal hypothalamus from adult male Wistar rats were processed by double label in situ hybridization using a 35S-labeled NPY complementary RNA probe and a digoxigenin-labeled GHR complementary RNA probe. In the medial basal hypothalamus, NPY messenger RNA (mRNA) was observed in the arcuate nucleus (ARC) and the dorsomedial nucleus. The majority (95%) of NPY mRNA-containing cells in the ARC expressed the GHR gene, whereas no NPY mRNA-containing cells in the dorsomedial nucleus expressed the GHR gene. These findings suggest that NPY neurons in the ARC mediate the feedback effect of GH on the hypothalamus.

Animals↗

Expression of proopiomelanocortin and prohormone convertase-1 and -2 in the late gestation fetal sheep pituitary.

The biological activity of fetal plasma immunoreactive ACTH has been reported to increase during the final weeks of gestation in fetal sheep, indicative of enhanced processing of POMC to ACTH. The present study was aimed at examining the expression and localization of the prohormone convertases, PC1 and PC2, in the pituitary of fetal sheep during the final weeks of gestation. Pituitaries were obtained from fetal sheep during the final 50 days gestation (dGA) at 100-107 dGA (n = 6), 117-121 dGA (n = 6), 126-130 dGA (n = 7), and 144-147 dGA (n = 8; term = approximately 148 dGA). Pituitaries were cryosectioned and subjected to dual labeling in situ hybridization using 35S-labeled PC1 and/or PC2 complementary RNA probes with a digoxigenin-labeled POMC complementary RNA to localize and quantify PC1 and PC2 messenger RNA (mRNA) in POMC-hybridizing cells. Immunocytochemistry was also performed to assess coexpression of PC1 and PC2 with ACTH in the fetal pituitary. PC1 mRNA was heterogeneously distributed in the anterior pituitary (AP) at all gestational ages examined, with hybridization signals observed over POMC-expressing cells (corticotropes) as well as over noncorticotrope phenotypes. The inferior region of the AP contained an approximately 3-fold greater (P < 0.01) percentage of POMC cells containing PC1 transcripts compared with the superior region of the AP. The proportion of POMC cells containing PC1 was significantly higher (P < 0.01) in the 100-107 dGA and 144-147 dGA groups than in the 117-121 dGA and 126-130 dGA groups in both inferior and superior AP. The intensity of the PC1 hybridization signal over POMC-expressing cells was also about 2- to 4-fold greater (P < 0.01) in the inferior compared with the superior region of the fetal AP; the intensity of the PC1 hybridization signal associated with POMC cells remained constant within the AP region and did not change over the gestational ages examined. Hybridization for PC1 was highly variable over regions of AP not hybridizing for POMC, probably due to differences in the level of mRNA for PC1 between phenotypes. Similar to POMC cells, the average hybridization signal for PC1 over non-POMC-hybridizing regions was about 2-fold greater in the inferior vs. superior AP. A weak PC2 hybridization signal was observed over a small number of unidentified phenotypes in the fetal AP at all ages examined; no POMC cells were found to contain PC2 hybridization signal. In the neurointermediate lobe, POMC, PC1, and PC2 were ubiquitously expressed at all ages. Levels of PC1 and PC2 mRNA in the fetal neurointermediate lobe did not change over the period of gestation examined. Immunocytochemical analysis of PC1 and PC2 with ACTH confirmed the pattern of expression and the extent of coexpression observed with in situ hybridization methods. We conclude that both PC1 and PC2 are likely to contribute to POMC processing in the fetal pituitary during the final weeks of gestation.

Animals↗