Transcript Isoform-Specific Estimation of Poly(A) Tail Length by Nanopore Sequencing of Native RNA.

The poly(A) tail is a homopolymeric stretch of adenosine at the 3'-end of mature RNA transcripts and its length plays an important role in nuclear export, stability, and translational regulation of mRNA. Existing techniques for genome-wide estimation of poly(A) tail length are based on short-read sequencing. These methods are limited because they sequence a synthetic DNA copy of mRNA instead of the native transcripts. Furthermore, they can identify only a short segment of the transcript proximal to the poly(A) tail which makes it difficult to assign the measured poly(A) length uniquely to a single transcript isoform. With the introduction of native RNA sequencing by Oxford Nanopore Technologies, it is now possible to sequence full-length native RNA. A single long read contains both the transcript and the associated poly(A) tail, thereby making transcriptome-wide isoform-specific poly(A) tail length assessment feasible. We developed tailfindr-an R-based package for estimating poly(A) tail length from Oxford Nanopore sequencing data. In this chapter, we describe in detail the pipeline for transcript isoform-specific poly(A) tail profiling based on native RNA Nanopore sequencing-from library preparation to downstream data analysis with tailfindr.

Evidence of an Unusual Poly(A) RNA Signature Detected by High-Throughput Chemical Mapping.

Homopolymeric adenosine RNA plays numerous roles in both cells and noncellular genetic material. We report herein an unusual poly(A) signature in chemical mapping data generated by the Eterna Massive Open Laboratory. Poly(A) sequences of length seven or more show unexpected results in the selective 2'-hydroxyl acylation read out by primer extension (SHAPE) and dimethyl sulfate (DMS) chemical probing. This unusual signature first appears in poly(A) sequences of length seven and grows to its maximum strength at length ∼10. In a long poly(A) sequence, the substitution of a single A by any other nucleotide disrupts the signature, but only for the 6 or so nucleotides on the 5' side of the substitution.

Poly(A) inclusive RNA isoform sequencing (PAIso-seq) reveals wide-spread non-adenosine residues within RNA poly(A) tails.

Message RNA poly(A) tails are vital for their function and regulation. However, the full-length sequence of mRNA isoforms with their poly(A) tails remains undetermined. Here, we develop a method at single-cell level sensitivity that enables quantification of poly(A) tails along with the full-length cDNA while reading non-adenosine residues within poly(A) tails precisely, which we name poly(A) inclusive RNA isoform sequencing (PAIso-seq). Using this method, we can quantify isoform specific poly(A) tail length. More interestingly, we find that 17% of the mRNAs harbor non-A residues within the body of poly(A) tails in mouse GV oocytes. We show that PAIso-seq is sensitive enough to analyze single GV oocytes. These findings will not only provide an accurate and sensitive tool in studying poly(A) tails, but also open a door for the function and regulation of non-adenosine modifications within the body of poly(A) tails.

A novel genome-wide polyadenylation sites recognition system based on condition random field.

Polyadenylation including the cleavage of pre-mRNA and addition of a stretch of adenosines to the 3'-end is an essential step of pre-mRNA processing in eukayotes. The known regulatory role of polyadenylation in mRNA localization, stability, and translation and the emerging link between poly(A) and disease states underline the necessary to fully characterize polyadenylation sites. Several artificial intelligence methods have been proposed for poly(A) sites recognition. However, these methods are suitable to small subsets of genome sequences. It is necessary to propose a method for genome-wide recognition of poly(A) sites. Recent efforts have found a lot of poly(A) related factors on DNA level. Here, we proposed a novel genome-wide poly(A) recognition method based on the Condition Random Field (CRF) by integrating multiple features. Compared with the polya_svm (the most accurate program for prediction of poly(A) sites till date), our method had a higher performance with the area under ROC curve(0.8621 versus 0.6796). The result suggests that our method is an effective method in genome wide poly(A) sites recognition.

E2F mediates enhanced alternative polyadenylation in proliferation.

BACKGROUND: The majority of mammalian genes contain multiple poly(A) sites in their 3' UTRs. Alternative cleavage and polyadenylation are emerging as an important layer of gene regulation as they generate transcript isoforms that differ in their 3' UTRs, thereby modulating genes' response to 3' UTR-mediated regulation. Enhanced cleavage at 3' UTR proximal poly(A) sites resulting in global 3' UTR shortening was recently linked to proliferation and cancer. However, mechanisms that regulate this enhanced alternative polyadenylation are unknown. RESULTS: Here, we explored, on a transcriptome-wide scale, alternative polyadenylation events associated with cellular proliferation and neoplastic transformation. We applied a deep-sequencing technique for identification and quantification of poly(A) sites to two human cellular models, each examined under proliferative, arrested and transformed states. In both cell systems we observed global 3' UTR shortening associated with proliferation, a link that was markedly stronger than the association with transformation. Furthermore, we found that proliferation is also associated with enhanced cleavage at intronic poly(A) sites. Last, we found that the expression level of the set of genes that encode for 3'-end processing proteins is globally elevated in proliferation, and that E2F transcription factors contribute to this regulation. CONCLUSIONS: Our results comprehensively identify alternative polyadenylation events associated with cellular proliferation and transformation, and demonstrate that the enhanced alternative polyadenylation in proliferative conditions results not only in global 3' UTR shortening but also in enhanced premature cleavage in introns. Our results also indicate that E2F-mediated co-transcriptional regulation of 3'-end processing genes is one of the mechanisms that links enhanced alternative polyadenylation to proliferation.

The human Pat1b protein: a novel mRNA deadenylation factor identified by a new immunoprecipitation technique.

The complex of the yeast Lsm1p-7p proteins with Pat1p is an important mRNA decay factor that is involved in translational shutdown of deadenylated mRNAs and thus prepares these mRNAs for degradation. While the Lsm proteins are highly conserved, there is no unique mammalian homolog of Pat1p. To identify proteins that interact with human LSm1, we developed a novel immunoprecipitation technique that yields virtually pure immunocomplexes. Mass-spec analysis therefore identifies mostly true positives, avoiding tedious functional screening. The method unambiguously identified the Pat1p homolog in HeLa cells, Pat1b. When targeted to a reporter mRNA, Pat1b represses gene expression by inducing deadenylation of the mRNAs. This demonstrates that Pat1b, unlike yPat1p, acts as an mRNA-specific deadenylation factor, highlighting the emerging importance of deadenylation in the mRNA regulation of higher eukaryotes.

Polyadenylation of ribosomal RNA in human cells.

The addition of poly(A)-tails to RNA is a process common to almost all organisms. In eukaryotes, stable poly(A)-tails, important for mRNA stability and translation initiation, are added to the 3' ends of most nuclear-encoded mRNAs, but not to rRNAs. Contrarily, in prokaryotes and organelles, polyadenylation stimulates RNA degradation. Recently, polyadenylation of nuclear-encoded transcripts in yeast was reported to promote RNA degradation, demonstrating that polyadenylation can play a double-edged role for RNA of nuclear origin. Here we asked whether in human cells ribosomal RNA can undergo polyadenylation. Using both molecular and bioinformatic approaches, we detected non-abundant polyadenylated transcripts of the 18S and 28S rRNAs. Interestingly, many of the post-transcriptionally added tails were composed of heteropolymeric poly(A)-rich sequences containing the other nucleotides in addition to adenosine. These polyadenylated RNA fragments are most likely degradation intermediates, as primer extension (PE) analysis revealed the presence of distal fragmented molecules, some of which matched the polyadenylation sites of the proximal cleavage products revealed by oligo(dT) and circled RT-PCR. These results suggest the presence of a mechanism to degrade ribosomal RNAs in human cells, that possibly initiates with endonucleolytic cleavages and involves the addition of poly(A) or poly(A)-rich tails to truncated transcripts, similar to that which operates in prokaryotes and organelles.

Primary effect of chemotherapy on the transcription profile of AIDS-related Kaposi's sarcoma.

BACKGROUND: Drugs & used in anticancer chemotherapy have severe effects upon the cellular transcription and replication machinery. From in vitro studies it has become clear that these drugs can affect specific genes, as well as have an effect upon the total transcriptome. METHODS: Total mRNA from two skin lesions from a single AIDS-KS patient was analyzed with the SAGE (Serial Analysis of Gene Expression) technique to assess changes in the transcriptome induced by chemotherapy. SAGE libraries were constructed from material obtained 24 (KS-24) and 48 (KS-48) hrs after combination therapy with bleomycin, doxorubicin and vincristine. KS-24 and KS-48 were compared to SAGE libraries of untreated AIDS-KS, and to libraries generated from normal skin and from isolated CD4+ T-cells, using the programs USAGE and HTM. SAGE libraries were also compared with the SAGEmap database. RESULTS: In order to assess the primary response of AIDS-related Kaposi's sarcoma (AIDS-KS) to chemotherapy in vivo, we analyzed the transcriptome of AIDS-KS skin lesions from a HIV-1 seropositive patient at two time points after therapy. The mRNA profile was found to have changed dramatically within 24 hours after drug treatment. There was an almost complete absence of transcripts highly expressed in AIDS-KS, probably due to a transcription block. Analysis of KS-24 suggested that mRNA pool used in its construction originated from poly(A) binding protein (PABP) mRNP complexes, which are probably located in nuclear structures known as interchromatin granule clusters (IGCs). IGCs are known to fuse after transcription inhibition, probably affecting poly(A)+RNA distribution.Forty-eight hours after chemotherapy, mRNA isolated from the lesion was largely derived from infiltrating lymphocytes, confirming the transcriptional block in the AIDS-KS tissue. CONCLUSIONS: These in vivo findings indicate that the effect of anti-cancer drugs is likely to be more global than up- or downregulation of specific genes, at least in this single patient with AIDS-KS. The SAGE results obtained 24 hrs after chemotherapy can be most plausibly explained by the isolation of a fraction of more stable poly(A)+RNA.

Genomic organization and promoter analysis of human KCNN3 gene.

KCNN3 is a member of the gene family, KCNN1-4, encoding the small and intermediate conductance calcium-activated potassium channels. Long CAG-repeat alleles of this gene have been found to be over-represented in patients with schizophrenia in a number of population-based association studies, and this gene maps to human chromosome 1q21, a region recently implicated in schizophrenia by linkage. To set the stage for a further functional evaluation of KCNN3, we defined the nature of the genomic locus in the size, structure, and sequence of its introns and exons and the function of potential upstream regulatory regions. We isolated P1-derived artificial chromosome (PAC) clones from a genomic library and identified an overlapping available bacterial artificial chromosome (BAC) clone. Cosmids subcloned from the PAC and BAC clones were then sequenced and merged with the sequence in the public database. The KCNN3 gene spans over 163.1 kb and is composed of eight exons and seven introns. All of the exon-intron junctions conform closely to consensus splice sites. The proximal 2.5 kb of the 5'-flanking sequence was obtained and analyzed for potential transcription factor binding sites. In the proximal 2.5 kb upstream region, potential sites for the Ikaros factor (IK2), homeodomain factor Nkx-2.5/Csx (NKX25), nuclear factor of activated T-cells (NFAT), upstream stimulating factor (USF), c-AMP responsive element binding protein (CREB), POU factor Brn2 (BRN-2), myeloid zinc finger protein (MZF1), vitellogenin binding protein (VBP), HNF3 forkhead homologue 2 (HFH2), and transcription initiation were identified, as well as several potential AP-1 and AP-4 sites. Finally, a 2261-bp fragment of this upstream region was cloned into a promoterless pGL3-luciferase vector, where it produced orientation-dependent expression of the reporter gene in transiently transfected PC12 cells, cells which natively express functional KCNN3 channels, suggesting that this cloned fragment includes competent promoter elements of this gene.

A new G-tailing method for the determination of the poly(A) tail length applied to hepatitis A virus RNA.

To study the role of the poly(A) tail length during the replication of poly(A)-containing plus-strand RNA virus, we have developed a simple reverse transcription polymerase chain reaction (RT-PCR)-based method that substantially improves the previously reported PAT [poly(A) test] assay. In contrast to the PAT assay, the new method is based on the enzymatic 3' elongation of mRNA with guanosine residues, thus immediately preserving the 3' end of the RNA and creating a unique poly(A)-oligo(G) junction. The oligo(G)-protected full-length poly(A) tail is reverse transcribed using the universal anti-sense primer oligo(dC(9)T(6)) and amplified by PCR with a gene-specific sense primer. After sequencing the resulting RT-PCR product the length of the poly(A) tail was unequivocally deduced from the number of adenosine residues between the oligo(G) stretch and the sequence upstream of the poly(A) tail. The efficiency and specificity of the newly developed assay was demonstrated by analysing the poly(A) tail length of the hepatitis A virus (HAV) RNA. We show here that the poly(A) tail of HAV RNA rescued after transfection of in vitro transcripts was elongated in the course of HAV replication.

Targeted disruption of the beta adducin gene (Add2) causes red blood cell spherocytosis in mice.

Adducins are a family of cytoskeleton proteins encoded by three genes (alpha, beta, gamma). In a comprehensive assay of gene expression, we show the ubiquitous expression of alpha- and gamma-adducins in contrast to the restricted expression of beta-adducin. beta-adducin is expressed at high levels in brain and hematopoietic tissues (bone marrow in humans, spleen in mice). To elucidate adducin's role in vivo, we created beta-adducin null mice by gene targeting, deleting exons 9-13. A 55-kDa chimeric polypeptide is produced from the first eight exons of beta-adducin and part of the neo cassette in spleen but is not detected in peripheral RBCs or brain. beta-adducin null RBCs are osmotically fragile, spherocytic, and dehydrated compared with the wild type, resembling RBCs from patients with hereditary spherocytosis. The lack of beta-adducin in RBCs leads to decreased membrane incorporation of alpha-adducin (30% of normal) and unexpectedly promotes a 5-fold increase in gamma-adducin incorporation into the RBC membrane skeleton. This study demonstrates adducin's importance to RBC membrane stability in vivo.

Human renin mRNA stability is increased in response to cAMP in Calu-6 cells.

The human carcinoma-derived cell line Calu-6 has previously been demonstrated to endogenously express human renin (hREN) mRNA and to markedly increase steady-state hREN mRNA levels (100-fold after 24 hours) in response to analogues of cAMP and postreceptor activators of adenylyl cyclase such as forskolin. However, both transfection analysis using hREN promoter-reporter constructs and nuclear run-on experiments suggest that transcriptional activity alone cannot account for this level of induction. We performed primer extension, reverse transcription-polymerase chain reaction, and 3' rapid amplification of cDNA ends to compare hREN mRNA between unstimulated and forskolin-stimulated cells. We demonstrate that hREN mRNA is identical under both conditions with respect to (1) utilization of the appropriate transcription start site, (2) processing of renin mRNA, and (3) utilization of the proper polyadenylation site and length of the poly-A tail. To address the mechanism of induction caused by cAMP, we used transcriptional inhibition and measured decay of hREN mRNA before and after forskolin or phorbol ester treatment. Experiments with both actinomycin D and 5, 6-dichlororibofuranosylbenzimidazole (DRB) showed that forskolin treatment markedly stabilized hREN mRNA in Calu-6 cells. A 2.3-fold increase in hREN mRNA half-life was also observed after treatment of Calu-6 cells with phorbol ester. Experiments with DRB demonstrated a similar robust stabilization of hREN mRNA after forskolin and phorbol ester treatment. These data demonstrate that the induction in hREN mRNA in response to both cAMP and phorbol ester occurs by a mechanism involving a posttranscriptional component.

Inosine exists in mRNA at tissue-specific levels and is most abundant in brain mRNA.

The general view that mRNA does not contain inosine has been challenged by the discovery of adenosine deaminases that act on RNA (ADARs). Although inosine monophosphate (IMP) cannot be detected in crude preparations of nucleotides derived from poly(A)+ RNA, here we show it is readily detectable and quantifiable once it is purified away from the Watson-Crick nucleotides. We report that IMP is present in mRNA at tissue-specific levels that correlate with the levels of ADAR mRNA expression. The amount of IMP present in poly(A)+ RNA isolated from various mammalian tissues suggests adenosine deamination may play an important role in regulating gene expression, particularly in brain, where we estimate one IMP is present for every 17 000 ribonucleotides.

Analysis of a polyadenine tract of the transforming growth factor-beta type II receptor gene in colorectal cancers by non-gel-sieving capillary electrophoresis.

We developed a method to analyze a polyadenine tract, the (A)10 repeat, within the cysteine-rich domain of the transforming growth factor-beta (TGF-beta) type II receptor gene using a non-gel-sieving capillary electrophoresis technique and applied it to the DNA diagnosis of colorectal cancers. This method consists of single-strand DNA amplification of the (A)10 repeat by an asymmetric PCR technique and capillary electrophoresis. A higher concentration of dATP in the PCR reaction mixture led to more specific amplification of the (A)10 repeat. Under the optimal electrophoretic conditions, one nucleotide difference could be determined in 8 to 32 nucleotides. One or two base deletions of the (A)10 repeat in colorectal cancers could be detected under these conditions within 30 min, and the results coincided with those obtained on DNA sequencing analyses. According to a sensitivity study, we could detect the deleted sequence if it was present in 12.5% or more of the wild-type allele. The reproducibility of this technique was satisfactory because the intraassay imprecision (CV) (n = 10) was 1.4%. These results indicate that capillary electrophoretic analysis of small repeated sequences results in easier handling and more feasible automation, compared with conventional gel electrophoretic analysis.

Post-transcriptional regulation of the gonadotropin-releasing hormone gene in GT1-7 cells.

GT1-7 cells respond to treatment with the phorbol ester, phorbol 12-myristate 13-acetate (PMA), with an inhibition of transcription of the proGnRH gene and decreases in GnRH mRNA levels. However, the timing of this decrease in GnRH mRNA levels suggests that a decrease in GnRH mRNA stability may be involved in addition to an inhibition of transcription of the proGnRH gene. To address this possibility, we treated GT1-7 cells with 100 nM PMA for 4 h and then monitored GnRH mRNA levels over time after blockade of GnRH gene transcription with DRB. PMA treatment caused GnRH mRNA half-life to decrease from 30 to 11 h. Then, to verify this observation, we examined changes in GnRH mRNA poly (A) tail length, which may be a reflection of mRNA turnover, following treatment of GT1-7 cells with PMA or vehicle for 0, 4, 8 or 24 h. The poly (A) tail was removed from half of the GT1 cytoplasmic RNA sample by digestion with RNase H and the difference in GnRH mRNA size with and without RNase H treatment was determined by Northern hybridization. PMA treatment (4 and 8 h) resulted in a significant decrease in the length of the GnRH mRNA poly (A) tail, consistent with a decrease in GnRH mRNA stability. This finding suggests that GnRH mRNA turnover is inducible by substances such as PMA. Our study indicates that a change in mRNA stability is one of a multiplicity of levels at which GnRH gene expression is regulated.

Effect of ovariectomy on the local residual ridge remodeling.

STATEMENT OF PROBLEM: Osteoporosis and edentulism are two disease processes that affect a large group of elderly people in the United States (24 and 25 million, respectively). These two diseases are independent of each other; however, they have several pathologic symptoms in common, such as reduction in bone mass. PURPOSE: The purpose of this study was to determine whether estrogen deficiency or its replacement therapy have any effect on the phenomenon of residual ridge remodeling. MATERIAL AND METHODS: Three animal groups were formed that consisted of six female Sprague-Dawley rats each. The two groups had ovariectomy and received either a vehicle solution or a daily dose (1.5 micrograms/day) of 17 beta-estradiol delivered through osmotic pumps. The control group underwent sham surgery and received a vehicle solution. Animals were pair fed throughout the experiment. Unilateral molar extraction was performed in the maxilla, which produced a suitable site for examination of histologic characteristics and molecular biologic analyses. At the 4-week postextraction period the bone remodeling activity was noted at the surface of the residual ridge in the control group. RESULTS: The ovariectomized group showed increased bone resorption activity, whereas the surface of the residual ridge alveolar bone of the ovariectomized and estrogen-treated group was covered by a layer of hyaline tissue. Poly(A)+ ribonucleic acid samples were isolated from the remodeling residual ridge tissues. Expression of alpha 2(I), alpha 1(II), alpha 1(IX), and alpha 2(X) collagens were examined by ribonucleic acid transfer dot blots. Compared with the control group, ovariectomized animals showed a reduction in bone formation with decreased expressions of type I and II collagens. In contrast, the estrogen-treatment group showed decreased formation of type I collagen with a much increased expression of type II collagen. Further examination of type II collagen formation on the ovariectomized and estrogen-treated group by means of in situ hybridization revealed the notable labeling by the type IIA collagen probe, which was associated with the surface tissue of the residual ridge alveolar bone. CONCLUSION: These findings suggest that estrogen deficiency and its replacement therapy seem to affect the activity of residual ridge bone remodeling at the molecular level.

Senescent heart compared with pressure overload-induced hypertrophy.

Although systolic left ventricular (LV) function is normal in the elderly, aging is associated in rat papillary muscle with mechanical and sarcoplasmic reticulum Ca2+ ATPase alterations similar to those observed in the hypertrophied heart. However, alterations in the other calcium-regulating proteins implicated in contraction and relaxation are still unknown. To investigate alterations in LV function and calcium-regulating proteins, we measured hemodynamics and Na(+)-Ca2+ exchanger (NCx), ryanodine receptor (RyR2), and sarcoplasmic reticular Ca2+ ATPase (SERCA2) mRNA levels (expressed in densitometric scores normalized to that of poly(A+) mRNA) in left ventricle from 4-month-old (adult, n = 13) and 24-month-old (senescent, n = 15) rats. For ex vivo contractile function, active tension was measured during isolated heart perfusion in adult (n = 11) and senescent (n = 11) rats. For comparison of age-dependent effects of moderate hypertension on both hemodynamics and calcium proteins, renovascular hypertension was induced or a sham operation performed at 2 (n = 11 and n = 6) and 22 (n = 26 and n = 5) months of age. In senescent rats, LV systolic pressure and maximal rates of pressure development were unaltered, although active tension was depressed (4.7 +/- 0.4 versus 8.3 +/- 0.7 g/g heart weight in adults, P < .0001). SERCA2 mRNA levels were decreased in senescent left ventricle (0.98 +/- 0.05 versus 1.18 +/- 0.05 in adults, P < .01), without changes in NCx and RyR2 mRNA accumulation. Renovascular hypertension resulted in 100% mortality in aged rats; in adults, renovascular hypertension resulted, 2 months later, in an increase of LV systolic pressure (170 +/- 7 versus 145 +/- 3 mm Hg in sham-operated rats, P < .05) and in mild LV hypertrophy (+18%, P < .01) associated with a decrease in SERCA2 mRNA levels (1.02 +/- 0.03 versus 1.18 +/- 0.03 in sham-operated rats, P < .001). Contractile dysfunction in senescent isolated heart and decreased SERCA2 mRNA levels were associated with in vivo normal LV function at rest, indicating the existence of in vivo compensatory mechanisms. RyR2 and NCx gene expressions were not implicated in the observed contractile dysfunction. In aged rats, renovascular hypertension resulted in 100% mortality, probably related to elevated levels of circulating angiotensin II, whereas in adult rats, renovascular hypertension induced a mild LV hypertrophy associated with a selective alteration in SERCA2 gene expression.

Development of an in vitro mRNA decay system for Escherichia coli: poly(A) polymerase I is necessary to trigger degradation.

Using a novel Escherichia coli in vitro decay system in which polysomes are the source of both enzymes and mRNA, we demonstrate a requirement for poly(A) polymerase I (PAP I) in mRNA turnover. The in vitro decay of two different mRNAs (trxA and lpp) is triggered by the addition of ATP only when polysomes are prepared from s strain carrying the wild-type gene for PAP I (pcnB+). The relative decay rates of these two messages are similar in vitro and in vivo. Poly(A) tails are formed on both mRNAs, but no poly(A) are detected on the 3' end of mature 23S rRNA. The size distribution of poly(A) tails generated in vitro, averaging 50 nt in length, is comparable to that previously reported in vivo. PAP I activity is associated exclusively with the polysomes. Exogenously added PAP I does not restore mRNA decay to PAP I-polysomes, suggesting that, in vivo, PAP I may be part of a multiprotein complex. The potential of this in vitro system for analyzing mRNA decay in E. coli is discussed.

In situ localization of cell synthesis and proliferation in periodontitis gingiva and tonsillar tissue.

OBJECTIVE: Previous work indicates that large numbers of B and T cells accumulate in the periodontal soft tissues although we know little about cellular synthetic activity and proliferation in this site. The aim of this study was to examine lymphocytic cell synthetic activity and proliferation in periodontitis gingiva and compare this to a known site of leucocyte proliferation, namely the oropharyngeal tonsils. MATERIALS AND METHODS: Messenger RNA (mRNA) and 28S ribosomal (28S rRNA) expressing cells in formalin-fixed/paraffin-embedded gingival and tonsillar tissue sections were detected by in situ hybridisation (ISH) using poly-deoxyribothymidine and 28S probes respectively. In addition S-phase proliferating and cycling cells were also detected by ISH with histone probes and by Ki-67 immunohistochemistry. Ten gingival biopsy samples were obtained from adult periodontitis patients and five tonsillar biopsies from tonsillectomy patients. RESULTS: Both mRNA and 28S rRNA-expressing cells were detected in all the samples tested. Plasma cells showed the strongest signal for the two probes and slight to moderate staining could be seen in epithelium, fibroblasts and endothelial cells. In contrast, gingival lymphocytes were either weakly stained or were unstained for these probes of synthetic activity. In tonsils, most lymphocytes in germinal centres showed moderate staining and mantol zone cells were much more weakly stained. In gingival samples, histone mRNA-expressing and cycling (Ki-67) cells were detected in 4/10, 10/10 cases respectively. These positive cells were mainly basal and suprabasal epithelial cells and a few mononuclear cells, whereas most germinal centre lymphocytes (B cells) were positive for this probe. The number of Ki67 positive cells was greater than histone mRNA bearing cells both in gingiva and tonsillar tissue. In contrast, mantol zone cells (mainly T cells) were sparsely stained by probes of cell proliferation. CONCLUSION: These results indicate that local proliferation of B cells does not occur in periodontitis gingiva in contrast with tonsillar tissue, although plasma cells showed strong synthetic activity in both tissues. T cells did not appear to proliferate greatly nor undergo active synthesis in either of these tissues. These findings substantiate previous hypotheses that specific leucocytes predominate in the gingival tissue through selective homing rather than by local proliferation.

The delay in rat liver regeneration by choline is associated to alteration in c-myc expression.

Previous data of our laboratory showed that female rats regenerated earlier than males and choline shifted the female growth pattern toward that of males. We investigated if the effect of choline on the liver compensatory growth was associated to a modulation of the expression and methylation pattern of an early cell cycle dependent proto-oncogene, c-myc. The peak of DNA synthesis was 22 h after 2/3 partial hepatectomy in female regenerating liver, while it was delayed to 30 h when female rats received choline for 3 weeks before liver surgery. Partial hepatectomy induced the expression of c-myc that was already maximal at 1 h. Choline reduced the c-myc expression and it shifted the maximum increase at 2 h. The methylation pattern of c-myc was studied with the Hpa II restriction enzyme. The delay in c-myc expression was not due to hypermethylation of the gene.