Evidences for a role of p38 MAP kinase in the stimulation of alkaline phosphatase and matrix mineralization induced by parathyroid hormone in osteoblastic cells.

Increased bone formation by PTH mainly results from activation of osteoblasts, an effect largely mediated by the cAMP-PKA pathway. Other pathways, however, are likely to be involved in this process. In this study we investigated whether PTH can activate p38 MAPK and the role of this kinase in osteoblastic cells. Bovine PTH(1-34) and forskolin markedly increased alkaline phosphatase (ALP) activity and doubled osteocalcin (Oc) expression in early differentiating MC3T3-E1 cells. These effects were associated with increase in cellular cAMP and activation of the MAP kinases ERK and p38. Activation of these MAP kinases was detectable after 1 h incubation with 10(-7) M PTH and lasted 1-2 h. Activation of p38 was mimicked by 10 microM forskolin and prevented by H89 suggesting a cAMP-PKA-dependent mechanism of p38 activation. Interestingly, PTH-induced ALP stimulation was dose-dependently inhibited by a specific p38 inhibitor with no change in the generation of cAMP and the production of osteocalcin. Similar inhibitory effect was obtained in cells stably expressing a dominant-negative p38 molecule. Finally, treatment of MC3T3-E1 cells with PTH for 3 weeks significantly enhanced matrix mineralization and this effect was markedly reduced by a selective p38 but not a specific MEK inhibitor. In conclusion, data presented in this study indicate that PTH can activate p38 in early differentiating osteoblastic cells. Activation of p38 is cAMP-PKA-dependent and mediates PTH-induced stimulation of ALP which plays a critical role for the calcification of the bone matrix.

Bone response to intermittent parathyroid hormone is altered in mice null for {beta}-Arrestin2.

Intermittent PTH administration increases bone turnover, resulting in net anabolic effects on bone. These effects are primarily mediated by intracellular cAMP signaling. However, the molecular mechanisms that regulate PTH activity in bone remain incompletely understood. beta-Arrestin2, a G protein-coupled receptor regulatory protein, inhibits PTH-stimulated cAMP accumulation in vitro. Using beta-arrestin2(-/-) (KO) and wild-type (WT) mice, we investigated the response to PTH in primary osteoblasts (POB) and the effects of intermittent PTH administration on bone mass and microarchitecture in vivo. Compared with that in WT mice, PTH-stimulated intracellular cAMP was increased and sustained in KO POB. Intermittent exposure of POB to PTH significantly decreased the ratio of osteoprotegerin (OPG) receptor activator of nuclear factor-kappaB ligand (RANKL) mRNA expression in KO POB, whereas it increased this ratio in WT POB. Total body bone mass and cortical and trabecular bone parameters were 5-10% lower in male KO mice compared with WT, and these differences were magnified upon in vivo administration of intermittent PTH (80 mug/kg.d) for 1 month. Thus, PTH significantly increased total body bone mineral content as well as vertebral trabecular bone volume and thickness in WT, but not KO mice. The anabolic response to PTH in cortical bone was also slightly more pronounced in WT than KO mice. Histomorphometry indicated that PTH prominently stimulated indexes of bone formation in both WT and KO mice, whereas it significantly increased indexes of bone resorption (i.e. osteoclast number and surface) in KO mice only. In conclusion, these results suggest that beta-arrestins may specify the activity of intermittent PTH on the skeleton by limiting PTH-induced osteoclastogenesis.

Vitamin D receptor gene start codon polymorphisms (FokI) and bone mineral density: interaction with age, dietary calcium, and 3'-end region polymorphisms.

Osteoporosis is a polygenic disease, whose determining loci have not yet been identified. Vitamin D receptor (VDR) gene polymorphisms in the 3'-end region (as determined by the enzymes BsmI and ApaI) have been inconsistently associated with bone mineral mass. More recently, VDR start codon polymorphisms (as determined by the enzyme FokI) have been found to be related to adult bone mineral density (BMD) in pre- and postmenopausal American women. We investigated the association between BMD and FokI genotypes in premenopausal European-Caucasian women as well as in prepubertal girls from the same genetic background and examined the interaction with VDR 3'-end region polymorphisms and with dietary calcium intake. Areal BMD (g/cm2) was measured by dual-energy X-ray absorptiometry at the level of the lumbar spine, femoral neck, and femoral shaft in 177 healthy premenopausal women (age range, 18.7-56.0 years) as well as in 155 prepubertal girls (age range, 6.6-11.4 years). Genotyping for FokI, BsmI, and ApaI VDR polymorphisms was performed using polymerase chain reaction methods. FokI genotype-dietary calcium interaction was cross-sectionally analyzed in all subjects and longitudinally in 103 prepubertal girls enrolled in a calcium intervention trial. The prevalence of FokI VDR gene polymorphisms in this cohort was 15% for ff, 50% for Ff, and 35% for FF. In the whole cohort of premenopausal women or prepubertal girls, no significant association was found between FokI VDR gene polymorphisms and BMD, even adjusted for age (Z score), weight, height, and calcium intake. Further analysis of FokI VDR gene polymorphisms and dietary calcium intake suggested a possible interaction in BMD determination, since a trend for an association with FokI genotypes was more evident at high than low calcium intake in both cross-sectional and longitudinal studies. Furthermore, cross-genotyping FokI and either BsmI or ApaI VDR polymorphisms suggested that the ff genotype was associated with a significantly lower lumbar spine BMD in bb and aa prepubertal girls. FokI VDR gene polymorphisms were not significantly associated with BMD in healthy European-Caucasian females. However, cross-genotyping of the VDR 3'-end and start codon polymorphic regions may provide a further insight into the complex determination of BMD.

Positive and negative control of the expression of parathyroid hormone (PTH)/PTH-related protein receptor via proximal promoter P3 in human osteoblast-like cells.

Three promoters, P1, P2, and P3, regulate the expression of the receptor for the human PTH/PTH -related protein. The P3 promoter, proximal to the gene, seems to be turned on in many tissues and to be the most active of the three in the human adult kidney. P3 is also active in human osteoblastic SaOS-2 cells. Its structure to function relationship is, however, still poorly understood. To address this issue we assayed, in transiently transfected SaOS-2 cells, the expression of reporter gene constructs containing truncated P3 promoter fragments and substitution mutants. We thus localized cis-acting elements essential for P3 promoter activity and identified two key Sp1 binding sites. We also found in the 5'-untranslated exon U4, transcribed from promoter P3, an element that inhibits the expression of the receptor and is not promoter specific. This study provides new insights into PTH receptor expression in human osteoblast-like cells.

Bone mineral mass and calcium and phosphate metabolism in young men: relationships with vitamin D receptor allelic polymorphisms.

The genetic bases of peak bone mass determinants are still poorly understood, particularly in males. We investigated the relationship between vitamin D receptor (VDR) 3'- and 5'-gene polymorphisms (as determined by the restriction enzymes BsmI and FokI) and bone mineral mass, and calcium and inorganic phosphate (Pi) metabolism in an homogeneous cohort of young healthy men. In 104 healthy subjects, aged 24.3 +/- 3.1 yr (mean +/- SD; range, 20.7-38.7), standardized bone mineral density (BMD; z-scores) at the levels of lumbar spine and femoral trochanter, i.e. bone mineral content adjusted for age, body size, and bone area, significantly differed between VDR 3'-end alleles (BsmI), whereas crude areal BMD or bone mineral content did not. Among BsmI homozygotes BB, BMD (z-scores) were significantly lower only in subjects also carrying the f allele at the VDR-5' polymorphic site (FokI). Serum PTH levels were significantly higher in the BB genotype at baseline and remained so under either a low or a high calcium-phosphorus diet. Moreover, on the low calcium-phosphorus diet, BB subjects had significantly decreased tubular Pi reabsorptive capacity and plasma Pi levels. Our results underline the importance of identifying multiple single base mutation polymorphisms within candidate genes of bone mineral mass and suggest a role for environmental/dietary factor interactions with VDR gene polymorphisms in peak bone mineral mass in men.

Structure of the murine Pit1 phosphate transporter/retrovirus receptor gene and functional characterization of its promoter region.

The Pit1 phosphate transporter (formerly also called Glvr-1) probably plays an important role in regulated phosphate handling in bone-forming cells. In this study, we describe the structure of the mouse Pit1 gene, as well as some functional characteristics of its promoter region in murine bone cells. Screening of a genomic library led to the isolation of two overlapping lambda clones containing 7kb of 5' flanking region, as well as the 10 exons of the mouse Pit1 gene corresponding to the published cDNA. The translation start site is located within exon I and the stop codon within exon X. The overall structure of the mouse gene is very similar to that of its human homolog, except for the presence of an additional 5' untranslated exon in human. The structure of the 5' untranslated region of the mouse gene was thus further investigated using rapid amplification of cDNA ends in murine ATDC5, MC3T3-E1 and Swiss 3T3 cells. The results indicate that, compared to the published cDNA, the mouse Pit1 gene contains in fact one additional 5' exon, which we named exon IA. Reporter gene assays demonstrate the presence of a functional TATA box containing promoter upstream of exon IA. This description of the murine Pit1 gene and of its promoter region paves the way to more detailed analyses concerning the regulation of Pit1 transcription in mouse cells. Furthermore, a comparison of mouse and human promoters will hopefully allow a better understanding of general mechanisms regulating Pit1 expression in different species.

A sensitive reporter gene system using bacterial luciferase based on a series of plasmid cloning vectors compatible with derivatives of pBR322.

We present a series of plasmid cloning vectors that are derived from a mutant of pSC101 possessing an elevated number of copies per genome equivalent. These vectors are compatible with any plasmid replicating from a pBR322 origin and use spectinomycin and/or streptomycin as a selective marker. They can be used whenever the simultaneous presence of several plasmids in the cell is desired. We use this vector system for the constitutive expression of the genes that are responsible for the production of the aldehyde substrate of bacterial luciferase. Transcription from promoters carried on a second plasmid can thus be measured within the living cell over a range of 3 orders of magnitude using bacterial luciferase as a reporter gene.

Species-specific mechanisms control the activity of the Pit1/PIT1 phosphate transporter gene promoter in mouse and human.

The Pit1 phosphate transporter is involved in regulated phosphate handling in bone forming cells. In this study, we compared the structure of the murine and human Pit1/PIT1 promoters and characterized cis-acting elements controlling Pit1/PIT1 expression. The Pit1/PIT1 promoter sequence and its location relative to the first transcribed exon are conserved and similar transcription factor binding sites are found at identical positions in mouse and human. Luciferase reporter gene assays in transiently transfected mouse ATDC5 chondrocytes and human SaOS-2 osteoblasts indicated that the activity of the mouse Pit1 promoter depends on several cis-acting elements, including ATF/CREB, Sp1 and AP-1 sites, an E-box and a TATA box. In contrast, the activity of the human promoter essentially requires a TATA-like sequence and one single Sp1 site. This Sp1 site binds Sp1, Sp3, as well as unidentified proteins present in SaOS-2 nuclear extracts and co-transfection experiments in SL2 cells indicate that Sp1 and Sp3 activate transcription from the human PIT1 promoter. These data suggest that, despite similarities in promoter structure, changes in the relative importance of conserved transcription factor binding sites cause species-dependent differences in Pit1 promoter function, which allow Sp1-related proteins to play a particularly important role in human.

Sequence and activity of parathyroid hormone/parathyroid hormone-related protein receptor promoter region in human osteoblast-like cells.

The parathyroid hormone (PTH)/PTH-related protein (PTHrP) receptor gene has been characterized in various species. The structure of its promoter and the regulation of its expression in human tissues have, however, not been clearly established yet. We characterized the region upstream of the PTH/PTHrP receptor gene and investigated its promoter activity in the human osteoblast-like SaOS-2 cells. In this region, three untranslated exons were localized, U1 and U2 by using a kidney cDNA, and U3 by homology with the mouse gene. In human osteoblast-like cells, a distal promoter (P1) was found to be inactive, as evaluated by luciferase reporter gene assays, in contrast to the situation found in human and mouse kidney tissue. A second promoter (P2), previously described in mouse and human kidney, was shown to be active in human osteoblast-like SaOS-2 cells. We found a hitherto uncharacterized promoter (P3), closely upstream of the ATG start codon. The activities of P2 and P3 were not additive. These results provide important information on the structure of the 5' flanking region of the human PTH/PTHrP gene receptor.

Characterization of the human Glvr-1 phosphate transporter/retrovirus receptor gene and promoter region.

The cell surface receptor for gibbon ape leukemia virus (Glvr-1) belongs to the type III sodium-dependent phosphate transporter/retrovirus receptor gene family. Several observations have suggested an important role for Glvr-1 in regulated Pi handling in bone forming cells and prompted us to investigate further the molecular mechanisms regulating Glvr-1 gene expression. In addition, the regulation of Glvr-1 gene expression also has potential applications to gene therapy, since retroviral vectors carrying gibbon ape leukemia virus envelope proteins are used for gene delivery into different cell types. The aim of this study was thus to clone the human Glvr-1 gene in order to describe its structure and its promoter region. Our results indicate that the Glvr-1 gene consists of 11 exons and 10 introns spread over 18kb of genomic DNA. The translation initiation site is located within exon II and the translation stop codon within exon XI. Rapid amplification of cDNA ends (5'-RACE) suggests that, in human SaOS-2 osteoblast-like cells, transcription of Glvr-1 is initiated at multiple sites, mostly located between bp 32 and 50 of the published cDNA sequence, which was initially obtained from HL-60 cells. The 5'-flanking region of the gene is characterized by a very high GC content. Reporter gene assays demonstrate the presence of a functional promoter upstream of exon I and indicate that a GC-rich region, containing two potential SP1 binding sites, is required for high promoter activity in transiently transfected SaOS-2 cells. The description of the human Glvr-1 gene structure, as well as the analysis of some structural and functional characteristics of its promoter region, provide a basis for more detailed investigation of the molecular mechanisms controlling expression of the Glvr-1 gene in bone forming cells and in other cell types.

Arginine increases insulin-like growth factor-I production and collagen synthesis in osteoblast-like cells.

Protein-energy malnutrition, which is common in elderly patients with osteoporotic hip fractures, is associated with reduced plasma levels of insulin-like growth factor-I (IGF-I). IGF-I is an important regulator of bone metabolism, particularly of osteoblastic bone formation both in vivo and in vitro. Pharmacological doses of arginine (Arg) increase growth hormone (GH) and IGF-I serum levels. Whether amino acids, particularly Arg, can directly modulate the production of IGF-I by osteoblasts is not known. We investigated the effects of increasing concentrations of Arg on IGF-I expression and production, alpha1(I) collagen expression and collagen synthesis, and cell proliferation and cell differentiation, as assessed by alkaline phosphatase (ALP) activity and osteocalcin (OC) release, in confluent mouse osteoblast-like MC3T3-E1 cells. The addition of Arg (7.5-7500 micromol/L, equivalent to 0.1- to 100-fold human plasma concentration) for 48 h increased IGF-I production (adjusted for cell number) in a concentration-dependent manner with a maximum of 2.3 +/- 0.3-fold at 7500 micromol/L Arg [x +/- standard error of the mean (SEM), n = 3 experiments, p < 0.01]. Arg (7.5-7500 micromol/L) increased the percentage of de novo collagen synthesis in a concentration-dependent manner (2.1 +/- 0.4-fold with 7500 micromol/L Arg, p < 0.001) and ALP activity with a maximal stimulation of 144% +/- 13% plateauing at 750 micromol/l Arg (p = 0.002). The steady state level of IGF-I messenger ribonucleic acid (mRNA) and alpha1(I) collagen mRNA (both normalized to cyclophilin mRNA) of cells incubated with Arg at high (100-fold) or low (0.1-fold) human plasma concentrations, was 1.4 +/- 0.2, 1.2 +/- 0.2, and 1.1 +/- 0.2 after 24 h for the 7.5, 1.8, and 0.9 kb IGF-I mRNA transcripts, respectively (n = 3 experiments) and 1.5 +/- 0.2 and 3.1 +/- 0.7 after 24 and 48 h, respectively, for the combined analysis of the 5.6 and 4.7 kb alpha1(I) collagen mRNA transcripts (n = 3 experiments). A maximal mitogenic effect (cell number) of +21% +/- 3% (p < 0.01) was obtained with 1000 micromol/L Arg. In contrast, Arg (7.5-7500 micromol/L) induced a reduction of OC production, which reached 30% +/- 3% with 7500 micromol/L Arg (p = 0.02). In conclusion, Arg stimulated IGF-I production and collagen synthesis in osteoblast-like cells. Thus, Arg may influence bone formation by enhancing local IGF-I production.

Functional importance of Myc-associated zinc finger protein for the human parathyroid hormone (PTH)/PTH-related peptide receptor-1 P2 promoter constitutive activity.

The aim of the present study was to analyze the functional importance for the parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor (PTHR1) gene P2 promoter activity of the putative proximal Myc-associated zinc finger protein (MAZ) site localized at position bp -45 to -39 bp, taking advantage of a G/A mutation identified at position -40 in the human sequence. Wild-type 'full-length' (1285P2) and truncated (760P2) promoter sequences were inserted upstream to the luciferase basic (pLucB) and enhancer (pLucE) reporter gene expression vectors. Transient transfections in osteoblast-like SaOS-2 cells and renal cells (RC.SV3A2) showed that the -40 G/A mutation significantly impaired transcriptional activity of wild-type 1285P2-pLucB and 760P2-pLucE promoter constructs. Further truncation of the P2 sequence demonstrated that the sequence -109/-37 bp was essential for promoter activity. Co-transfection with a MAZ expression vector did not modify the wild-type 1285P2-pLucB construct reporter activity but significantly increased 2-fold the mutated construction activity (P<0.05). Electrophoretic mobility shift assays using SaOS-2 nuclear extracts and a double-stranded DNA fragment encompassing the -45 to -39 putative MAZ site (ds-MAZ-oligo) disclosed two specific DNA-protein complexes. Complex II (fast moving) had a lower affinity for the mutated MAZ motif than for the wild-type MAZ motif while complex I (slow moving) had the same affinity for both wild-type or mutated MAZ sequences. Competition studies with Sp1 consensus oligonucleotide (ds-Sp1-oligo) markedly reduced complex I intensity, with a concomitant increase in that of complex II. Finally, ribonuclease protection assays showed that P2-specific PTHR1 mRNA transcript expression was significantly decreased in SaOS-2 cells transfected with ds-MAZ-oligo as compared with that for control (P<0.001) and ds-Sp1-oligo (P<0.05). Taken together, our studies suggest that the putative -45 to -39 MAZ-binding site regulates the constitutive activity of human PTHR1 P2 promoter.

Analysis of a copy number mutant of plasmid pSC101: co-maintenance of wild type and mutant plasmids.

We have isolated a high copy number mutant of plasmid pSC101 which is maintained at a level 4 times higher than that of the wild type. The mutation is a single base change that maps in codon 93 of the initiation protein RepA. We find that the mutation relaxes the autoregulation of the protein but increases its affinity for the repeated sequences in the origin. The wild type and the mutant repA genes are co-dominant and the mutated protein acts in trans even in the presence of the wild type protein. Co-maintenance of the two types of plasmids results in an intermediate copy number. Computer simulation indicates that simple models can explain the behaviour of the two plasmids.

Calcium stimulates parathyroid hormone-related protein production in Leydig tumor cells through a putative cation-sensing mechanism.

The production of parathyroid hormone-related protein (PTHrP) is regulated by a variety of hormones and growth factors. Previous research has shown that several PTHrP-producing cells are influenced by extracellular calcium (Ca(2+)(o)) concentration, with elevated levels increasing PTH-like activity released by cultured H500 rat Leydig tumor cells through a post-transcriptional mechanism. We have investigated the hypothesis that calcium stimulates PTHrP production in H500 cells by interacting with a cell membrane-associated cation-sensing receptor. Besides increased Ca(2+)(o) concentration, magnesium and the polycationic antibiotic neomycin also increased PTHrP production in a concentration-dependent manner. In the presence of the calcium ionophore, ionomycin, which markedly elevated cytosolic free calcium, the stimulation by Ca(2+)(o) of PTHrP could still be detected. These results indicate that increasing Ca(2+)(o) stimulates PTHrP production, possibly through a putative cell membrane-associated calcium-sensing mechanism. RT-PCR revealed the presence of a very small amount of calcium-sensing receptor coding mRNA.

The replication of plasmid pSC101.

The origin of replication of plasmid pSC101 presents features reminiscent of those found in a number of plasmids. As for those plasmids, many details about the way it initiates its replication are beginning to be known, but the regulation of this process will not be easily understood.

A locus involved in the regulation of replication in plasmid pSC101.

The origin of replication of plasmid pSC101 contains three directly repeated sequences RS1, RS2, and RS3 separated by 22 bp from two palindromic sequences, IR1 and IR2, which are partially homologous to the direct repeats. These inverted repeat (IR) sequences overlap the promoter of the repA gene which encodes a protein essential for plasmid replication. We have shown that RepA binds to the RS sites as a monomer and to the IR sites as a dimer. The influence of the IR1 site, and of the DNA segment that separates it from RS3, on plasmid copy number control has been studied in detail. We show that the integrity of IR1 is essential for efficient replication and plasmid stability, the critical site extending to the left of IR1 proper. We also show that the presence of IR1 modifies profoundly the binding properties of purified RepA protein to a segment of DNA containing the RS sequences. IR1 is separated from its homologous site on RS3 by approximately four turns of the DNA helix. Replication is abolished if this distance is increased by half a turn of the helix but it is restored if the distance is increased by a whole turn. These results suggest a DNA looping interaction, in the initiation of replication, between the RepA dimer that binds IR1 and the RepA monomers that bind the RS sequences.

The par region of pSC101 affects plasmid copy number as well as stability.

The par locus is a segment of pSC101 that has been identified as a cis-acting determinant of plasmid stability. We show that par also determines copy number and must, therefore, play a role in plasmid replication. The segregation defect, but not the copy-number reduction, of par- replication origins is completely suppressed by a short sequence from the bacteriophage lambda gene O which is present in plasmid pKO-4. Thus, replication and segregation functions are separable from each other.

Monomers and dimers of the RepA protein in plasmid pSC101 replication: domains in RepA.

The replication of plasmid pSC101 requires the plasmid-encoded protein RepA. This protein has a double role: it binds to three directly repeated sequences in the pSC101 origin and promotes replication of the plasmid; it binds to two inversely repeated sequences in its promoter region and regulates its own transcription. A series of RepA protein derivatives carrying deletions of the C-terminal region were assayed for specific binding. We found that the last third of the protein is not needed for binding to the various specific sites. Truncated proteins that still bind can also form heterodimers with a wild-type protein. Analysis of band retardation assays conducted with wild-type and truncated proteins indicates that RepA binds to directly repeated sequences as a monomer and to inversely repeated sequences as a dimer.

In vivo and in vitro studies of a copy number mutation of the RepA replication protein of plasmid pSC101.

The RepA replication protein of plasmid pSC101 binds as a monomer to three repeated sequences (RS1, RS2, and RS3) in the replication origin of the plasmid to initiate duplication and binds as a dimer to two inversely repeated sequences (IR1 and IR2) in its promoter region (D. Manen, L. C. Upegui-Gonzalez, and L. Caro, Proc. Natl. Acad. Sci. USA 89:8923-8927, 1992). The binding to IR2 autoregulates repA transcription (P. Linder, G. Churchward, G. X. Xia, Y. Y. Yu, and L. Caro, J. Mol. Biol. 181:383-393, 1985). A mutation in the protein RepA(cop) that affects a single amino acid increases the plasmid copy number fourfold. In vivo experiments show that, when provided in trans under a foreign promoter, the RepA(cop) protein increases the replication of a plasmid containing the origin of replication without repA, whereas it decreases the repression of its own promoter. In vitro experiments show that the purified RepA(cop) protein binds more efficiently to the repeated sequences within the origin than does RepA and that its binding to these sequences is more specific than that of RepA. Binding to an inversely repeated sequence within the repA promoter gives opposite results: the wild-type protein binds efficiently to that sequence, whereas the mutated protein binds less efficiently and less specifically. Footprint experiments confirmed these results and, in addition, showed a difference in the pattern of protection of the inversely repeated sequences by the mutant protein. Equilibrium binding experiments showed that the formation of protein-probe complexes at increasing concentrations of protein had a sigmoidal shape for binding to RS sequences and a hyperbolic shape for binding to IR sequences. The results, together with earlier work (G.-X. Xia, D. Manen, T. Goebel, P. Linder, G. Churchward, and L. Caro, Mol. Microbiol. 5:631-640, 1991), confirm that the binding of RepA to RS sequences plays a crucial role in the regulation of plasmid replication and that its binding to IR sequences plays a role in the autoregulation of RepA expression. They also demonstrate that the two separate functions of the protein are effected by two different forms of binding to the target sites.

Transcription events in the origin of replication of plasmid pSC101.

Insertion mutations were isolated in the origin fragment of the plasmid pSC101 after random cleavage with DNase I. The replication properties of the resulting plasmids confirmed previous findings and extended the characterization of the essential regions. Using these plasmids, we analyzed by various methods the transcription events in the pSC101 origin. In addition to the mRNA of repA, a gene coding for the self-regulated RepA protein which is essential for replication of the plasmid, we characterized a transcript, which we called RNA Y, that runs in the opposite direction and that starts in the middle of the second repeated sequence in the origin region. Like the self-regulated repA mRNA, RNA Y is weakly expressed. It does not code for a complete protein within the origin fragment but may do so in the wild-type plasmid. We also found indications for one or, possibly, two small RNA species, called RNA X, which run in the same direction as RepA and which are partially complementary to RNA Y. We postulate that RNA Y and, possibly, RNA X are implicated in the initiation of replication of pSC101.