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B Grimes

Publications and source records attributed to B Grimes.

At least 19 recordsLinked to original sources
PubMed2006

The isoflavone genistein inhibits LPS-stimulated TNFalpha, but not IL-6 expression in monocytes from hemodialysis patients and healthy subjects.

BACKGROUND: Whole blood and peripheral blood mononuclear cells from hemodialysis (HD) patients show increased production and secretion of inflammatory cytokines. We determined the contribution of blood monocytes to the production of inflammatory cytokines in whole blood from HD patients. METHODS: Whole blood and isolated mononuclear cells from HD patients and healthy control subjects were preincubated with the isoflavone genistein and stimulated with LPS. TNFalpha, IL-6 and IL-10 formation in the whole blood was measured with ELISA and intracellular cytokine formation in CD 14-positive monocytes was determined by flow cytometry. RESULTS: Unstimulated blood levels of TNFalpha, IL-6 and IL-10 were significantly elevated in HD patients compared to controls, but intracellular monocyte content of these cytokines was identical between groups. LPS induced a robust TNFalpha response in both whole blood and monocytes, and TNFalpha formation was 2.3-fold higher in blood from HD patients compared to controls. A similar trend was observed in monocytes. Conversely, LPS stimulation increased IL-6 levels >1000-fold in whole blood, albeit without a noticeable difference between groups. Only minor increases in monocyte IL-6 content were observed. The isoflavone genistein did not inhibit IL-6 formation and did not alter basal TNFalpha levels, but genistein selectively blocked LPS-induced TNFalpha formation in whole blood and monocytes from both groups. CONCLUSION: Intracellular levels of TNFalpha, IL-6 and IL-10 in monocytes are indistinguishable between HD patients and healthy controls. However, monocytes from HD patients are selectively primed for enhanced TNFalpha secretion in response to LPS. The selective inhibition of monocyte TNFalpha production by genistein may explain the anti-inflammatory action of this phytochemical observed in vivo.

Adult↗
PubMed2001

Nuclear factor-kappaB is constitutively activated in primitive human acute myelogenous leukemia cells.

Human acute myelogenous leukemia (AML) is thought to arise from a rare population of malignant stem cells. Cells of this nature, herein referred to as leukemic stem cells (LSCs), have been documented for nearly all AML subtypes and appear to fulfill the criteria for stem cells in that they are self-renewing and give rise to the cells found in many leukemic populations. Because these cells are likely to be critical for the genesis and perpetuation of leukemic disease, the present studies sought to characterize unique molecular properties of the LSC population, with particular emphasis on the transcription factor, nuclear factor-kappaB (NF-kappaB). Previous experiments have shown that unstimulated human CD34(+) progenitor cells do not express NF-kappaB. In contrast, primary AML CD34(+) cells display readily detectable NF-kappaB activity as assessed by electrophoretic mobility shift assay and gene expression studies. Furthermore, detailed analyses of enriched AML stem cells (CD34(+)/CD38(-)/CD123(+)) indicate that NF-kappaB is also active in the LSC population. Given the expression of NF-kappaB in leukemic, but not normal primitive cells, the hypothesis that inhibition of NF-kappaB might induce leukemia-specific apoptosis was tested by treating primary cells with the proteasome inhibitor MG-132, a well-known inhibitor of NF-kappaB. Leukemic CD34(+)/CD38(-) cells displayed a rapid induction of cell death in response to MG-132, whereas normal CD34(+)/CD38(-) cells showed little if any effect. Taken together, these data indicate that primitive AML cells aberrantly express NF-kappaB and that the presence of this factor may provide unique opportunities to preferentially ablate LSCs.

Actins↗
PubMed2001

Differential homing and engraftment properties of hematopoietic progenitor cells from murine bone marrow, mobilized peripheral blood, and fetal liver.

The rate of reconstitution following hematopoietic stem cell (HSC) transplantation differs widely depending on the tissue source of the cells infused. To test the hypothesis that variability in engraftment kinetics is related to differences in the efficiency with which intravenously transplanted HSCs "home" to the bone marrow (BM), the homing properties of murine fetal liver (FL), adult BM, and mobilized peripheral blood (MPB) cells were compared. Lethally irradiated mice transplanted with 2 x 10(6) FL, BM, or MPB cells exhibited sequentially slower recovery of circulating leukocytes and platelets that correlates with the progressively lower frequency of colony-forming cells (CFCs) in these tissues. However, differences in the rate and degree of early and long-term reconstitution were maintained even after infusing equal numbers of CFCs derived from FL, BM, and MPB. To compare the homing of progenitors from these tissues, cells were labeled with fluorescent PKH26 dye and injected into lethally irradiated hosts. Three hours later, PKH26(+) cells were reisolated from the BM and spleen by fluorescence-activated cell sorting and assayed for in vitro CFCs. Despite the higher level of very late antigen (VLA)-2, VLA-4, and VLA-5 on Sca-1(+)c-kit(+) cells from FL compared to BM, 10-fold fewer FL CFCs homed to hematopoietic organs than those from BM. MPB cells homed slightly better, but still less efficiently than BM cells. Therefore, clonogenic cells from different tissues exhibit striking variations in homing efficiency that does not necessarily correlate with engraftment kinetics. Homing is likely counterbalanced by intrinsic differences in proliferative potential that ultimately determine the rate of hematopoietic reconstitution.

Animals↗
PubMed2001

Expression of tumor-suppressor genes interferon regulatory factor 1 and death-associated protein kinase in primitive acute myelogenous leukemia cells.

Previous studies indicate that human acute myelogenous leukemia (AML) arises from a rare population of leukemic stem cells. Cells of this nature can initiate and maintain leukemic cell growth in both long-term cultures and nonobese diabetic/severe combined immune-deficient mice. To characterize the biology of primitive AML cells, gene expression screens were performed with 7 primary AML and 3 normal specimens. For each sample, stem cell populations (CD34(+)/CD38(-)) were isolated and used to synthesize radiolabeled complementary DNA (cDNA). AML vs normal probes were then hybridized to cDNA arrays containing genes related to cancer and apoptosis. Of approximately 1400 genes analyzed, 2 tumor-suppressor genes were identified that were overexpressed in all 7 of the AML CD34(+)/CD38(-) cell populations: death-associated protein kinase and interferon regulatory factor 1. Expression of each gene was confirmed by reverse-transcription polymerase chain reaction and immunoblot analysis. It is proposed that tumor-suppressor proteins play a role in the biology of primitive AML cells. (Blood. 2001;97:2177-2179)

ADP-ribosyl Cyclase↗
PubMed2001

Homing and engraftment defects in ex vivo expanded murine hematopoietic cells are associated with downregulation of beta1 integrin.

OBJECTIVE: Hematopoietic progenitors generated by ex vivo expansion "home" less efficiently to the bone marrow (BM) after intravenous transplantation than fresh cells. To explore the underlying cause of this transplantation defect, we examined the homing and engraftment properties in vivo of fresh and cultured marrow cells differing in beta1 integrin expression. MATERIALS AND METHODS: Fresh murine BM cells, or the expanded progeny of enriched Sca-1(+) c-kit(+)Lin(-) stem cells, were fractionated into beta1(-/lo) and beta1(+) subpopulations by cell sorting. These populations were assayed for their content of in vitro colony-forming cells (CFCs), cells able to provide radioprotection, and early and long-term multilineage hematopoietic reconstitution following transplantation into myeloablated recipients. These endpoints were correlated with the homing properties of beta1(-/lo) and beta1(+) cells that were labeled with 5- (and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE) and tracked to hematopoietic organs 24 hours after injection into lethally irradiated mice. RESULTS: Most normal stem and progenitor cells express high levels of beta1 integrin. In contrast, most clonogenic cells generated in vitro are beta1(-/lo). Consequently, expanded beta1(-/lo) progenitors failed to provide radioprotection or repopulate the hematopoietic system following intravenous transplantation. Defective engraftment of expanded cells was associated with reduced homing of beta1(-/lo) cells to the bone marrow. CONCLUSION: Downregulation of beta1 integrin on primitive hematopoietic cells during ex vivo expansion reduces their homing efficiency and negatively impacts hematopoietic reconstitution in vivo. Strategies directed at preserving beta1 integrin expression during culture may improve the clinical utility of expanded hematopoietic cells.

Animals↗
PubMed2000

Distinct functional properties of highly purified hematopoietic stem cells from mouse strains differing in stem cell numbers.

We have previously demonstrated that young adult DBA/2 (DBA) mice have more stem cells than C57BL/6 (B6) mice, as measured in a cobblestone area-forming cell (CAFC) assay using unfractionated marrow. To study the nature of this difference, we have now compared the proliferative fate of single, highly enriched Sca-1(+)c-kit(+)Lin(-) stem cells from these strains. Although equal in frequency, functional comparison revealed that Sca-1(+)c-kit(+)Lin(-) cells from DBA mice contained twice as many cells with CAFC activity. DBA clones persisted much longer in vitro, and developed later in time. To assess whether these differences were of any functional relevance in vivo, we compared engraftment of lethally irradiated mice transplanted with 1000 B6 or DBA Sca-1(+)c-kit(+)Lin(-) cells. Recipients of enriched DBA cells recovered much faster than animals transplanted with B6 cells. We also studied endogenous hematopoietic recovery after 5-fluorouracil (5-FU) treatment in vivo. Progenitors and peripheral blood cells recovered twice as fast in DBA mice. Thus, DBA stem cells have superior proliferative potential compared with phenotypically identical stem cells obtained from B6 mice. Such genetically determined quantitative and qualitative differences in stem cell behavior likely contribute to the dramatically different hematopoietic recovery rates observed in human transplant patients. (Blood. 2000;96:1374-1379)

Animals↗
PubMed2000

Mammalian artificial chromosome formation from circular alphoid input DNA does not require telomere repeats.

Mammalian artificial chromosomes (MACs) form in HT1080 cells after transfecting linear yeast artificial chromosome constructs minimally containing competent alphoid arrays, a selectable marker and terminal human telomere repeats. Restrictions on the structure of input DNA in MAC formation were investigated by transfecting circular or linear alphoid constructs with or without human telomere arrays and by varying the position and orientation of the telomere arrays on input linear constructs. Circular input DNA efficiently produced MACs. Absence of telomere arrays from circular input molecules did not significantly alter MAC formation rates. Linear constructs capped with telomere arrays generated MACs effectively, but a severe reduction in MAC formation was observed from linear constructs lacking telomere arrays. Human telomere arrays positioned 1-5 kb from linear construct ends and in either orientation were able to promote MAC formation with similar efficiencies. Both circular and linear input constructs generated artificial chromosomes that efficiently segregated in the absence of selection. Telomeres were not detected on the MACs, regardless of the inclusion of telomere arrays on input DNA, suggesting that circular chromosomes were formed. We found no evidence for acquisition of host cell DNA, which is consistent with de novo chromosome assembly.

Bacteriophage P1↗
PubMed2000

Effects of cell cycle activation on the short-term engraftment properties of ex vivo expanded murine hematopoietic cells.

Loss of long-term hematopoietic stem cell function in vitro is associated with cell cycle progression. To determine whether cytokine-induced proliferation also limits the rate of short-term engraftment and potential clinical utility of ex vivo expanded hematopoietic cells, murine Sca-1(+)c-kit(+)Lin(-) cells were cultured in interleukin-6 (IL-6), IL-11, granulocyte colony-stimulating factor (G-CSF), stem cell factor, flk-2 ligand, and thrombopoietin for 7 days. Cells amplified 2000-fold were then stained with Hoechst 33342, separated into G(0)/G(1) (72% +/- 3%) or S/G(2)/M (27% +/- 3%) fractions by flow sorting, and injected into lethally irradiated mice. Although long-term (more than 6 months) engraftment of lymphoid and myeloid lineages was greater in primary and secondary recipients of expanded cells residing in G(0)/G(1) at the time of transplantation, there were no noted differences in the short-term (less than 6 weeks) recovery kinetics of circulating blood cells. When hematopoietic cells were expanded in cultures containing the tetrapeptide stem cell inhibitor N-Acetyl-Ser-Asp-Lys-Pro (AcSDKP) to reduce progenitor cycling prior to transplantation, again there were no differences observed in short-term reconstitution by inhibited or uninhibited cells. Interestingly, AcSDKP significantly accelerated engraftment by expanded hematopoietic cells when administered in vivo at the time of transplantation. Leukocytes recovered to 20% of normal levels approximately 1 week faster, and thrombocytopenia was largely abrogated in AcSDKP-treated versus untreated mice. Therefore, while AcSDKP can accelerate the engraftment of ex vivo expanded hematopoietic progenitors, which suggests a relatively simple approach to improve their clinical utility, its effects appear unrelated to cell cycle arrest. (Blood. 2000;95:2829-2837)

Animals↗
PubMed2000

The interleukin-3 receptor alpha chain is a unique marker for human acute myelogenous leukemia stem cells.

Recent studies suggest that the population of malignant cells found in human acute myelogenous leukemia (AML) arises from a rare population of leukemic stem cells (LSCs). LSCs have been documented for nearly all AML subtypes and have been phenotypically described as CD34+/CD38- or CD34+/HLA-DR-. Given the potentially critical role of these primitive cells in perpetuating leukemic disease, we sought to further investigate their molecular and cellular characteristics. Flow cytometric studies using primary AML tissue showed that the interleukin-3 receptor alpha chain (IL-3Ralpha or CD123) was strongly expressed in CD34+/CD38- cells (98 +/- 2% positive) from 16 of 18 primary specimens. Conversely, normal bone marrow derived CD34+/CD38- cells showed virtually no detectable expression of the CD123 antigen. To assess the functional role of IL-3Ralpha positive cells, purified CD34+/CD123+ leukemia cells were transplanted into immune deficient NOD/SCID mice. These experiments showed that CD123+ cells were competent to establish and maintain leukemic populations in vivo. To begin to elucidate a biological role for CD123 in leukemia, primary AML samples were analyzed with respect to signal transduction activity in the MAPK, Akt, and Stat5 pathways. Phosphorylation was not detected in response to IL-3 stimulation, thereby suggesting CD123 is not active in conventional IL-3-mediated signaling. Collectively, these data indicate that CD123 represents a unique marker for primitive leukemic stem cells. Given the strong expression of this receptor on LSCs, we propose that targeting of CD123 may be a promising strategy for the preferential ablation of AML cells.

Animals↗
PubMed1999

A human DAZ transgene confers partial rescue of the mouse Dazl null phenotype.

In a subset of infertile men, a spectrum of spermatogenic defects ranging from a complete absence of germ cells (sertoli cell only) to oligozoospermia is associated with microdeletions of the DAZ (deleted in azoospermia) gene cluster on human distal Yq. DAZ encodes a testis-specific protein with RNA-binding potential recently derived from a single-copy gene DAZL1 (DAZ-like) on chromosome 3. Y chromosomal DAZ homologues are confined to humans and higher primates. It remains unclear which function unique to higher primate spermatogenesis DAZ may serve, and the functional status of the gene recently has been questioned. To assess the extent of functional conservation we have tested the capacity of a human DAZ gene contained in a 225-kb yeast artificial chromosome to complement the sterile phenotype of the Dazl null mouse (Dazl-/-), which is characterized by severe germ-cell depletion and meiotic failure. Although Dazl-/- mice remained infertile when the DAZ transgene was introduced, histological examination revealed a partial and variable rescue of the mutant phenotype, manifest as a pronounced increase in the germ cell population of the seminiferous tubules and survival to the pachytene stage of meiosis. As well as constituting definitive proof of the spermatogenic role of the DAZ gene product, these findings confirm the high degree of functional conservation between the DAZ and DAZL1 genes, suggesting they may constitute a single target for contraceptive intervention and raising the possibility of therapeutic up-regulation of the DAZL1 gene in infertile men.

Animals↗
PubMed1999

Organ-selective homing defines engraftment kinetics of murine hematopoietic stem cells and is compromised by Ex vivo expansion.

Hematopoietic reconstitution of ablated recipients requires that intravenously (IV) transplanted stem and progenitor cells "home" to organs that support their proliferation and differentiation. To examine the possible relationship between homing properties and subsequent engraftment potential, murine bone marrow (BM) cells were labeled with fluorescent PKH26 dye and injected into lethally irradiated hosts. PKH26(+) cells homing to marrow or spleen were then isolated by fluorescence-activated cell sorting and assayed for in vitro colony-forming cells (CFCs). Progenitors accumulated rapidly in the spleen, but declined to only 6% of input numbers after 24 hours. Although egress from this organ was accompanied by a simultaneous accumulation of CFCs in the BM (plateauing at 6% to 8% of input after 3 hours), spleen cells remained enriched in donor CFCs compared with marrow during this time. To determine whether this differential homing of clonogenic cells to the marrow and spleen influenced their contribution to short-term or long-term hematopoiesis in vivo, PKH26(+) cells were sorted from each organ 3 hours after transplantation and injected into lethally irradiated Ly-5 congenic mice. Cells that had homed initially to the spleen regenerated circulating leukocytes (20% of normal counts) approximately 2 weeks faster than cells that had homed to the marrow, or PKH26-labeled cells that had not been selected by a prior homing step. Both primary (17 weeks) and secondary (10 weeks) recipients of "spleen-homed" cells also contained approximately 50% higher numbers of CFCs per femur than recipients of "BM-homed" cells. To examine whether progenitor homing was altered upon ex vivo expansion, highly enriched Sca-1(+)c-kit+Lin- cells were cultured for 9 days in serum-free medium containing interleukin (IL)-6, IL-11, granulocyte colony-stimulating factor, stem cell factor, flk-2/flt3 ligand, and thrombopoietin. Expanded cells were then stained with PKH26 and assayed as above. Strikingly, CFCs generated in vitro exhibited a 10-fold reduction in homing capacity compared with fresh progenitors. These studies demonstrate that clonogenic cells with differential homing properties contribute variably to early and late hematopoiesis in vivo. The dramatic decline in the homing capacity of progenitors generated in vitro underscores critical qualitative changes that may compromise their biologic function and potential clinical utility, despite their efficient numerical expansion.

Animals↗
PubMed1999

Genetic manipulation of primitive leukemic and normal hematopoietic cells using a novel method of adenovirus-mediated gene transfer.

Gene transfer into early hematopoietic cells has been problematic due to the quiescent nature of primitive cells and the lack of gene transfer vehicles with high efficiency for hematopoietic cell types. Previously, we have shown that adenoviral vectors can be used for the transduction of normal human progenitors with gene transfer efficiencies of approximately 30%. However, this approach is limited by relatively slow uptake kinetics (24-48 h) and a strong dependence on the presence of exogenous cytokines. Thus, we have modified this approach by combining adenoviral vectors with polycations to generate a virus-polycation complex, or VPC. Vehicles of this nature, when composed of conventional adenoviral vectors and polyamidoamine dendrimers, are a highly efficient means of transducing both normal and acute myelogenous leukemia (AML) cells. Moreover, the kinetics of gene transfer are markedly increased using the VPC strategy, with approximately 70% of transduction complete within 2 h. In this study, using viruses that encode green fluorescence protein (GFP), or the T cell costimulatory molecule B7.1 (CD80), we show that VPC-mediated gene transfer is an effective means of transducing normal and AML cells, including those with a highly primitive phenotype. Our data suggest that transient genetic manipulation of primitive hematopoietic cells can readily be achieved and should therefore permit a variety of research and clinical endeavors.

Acute Disease↗
PubMed1998

Assay of centromere function using a human artificial chromosome.

In order to define a functional human centromere sequence, an artificial chromosome was constructed as a reproducible DNA molecule. Mammalian telomere repeats and a selectable marker were introduced into yeast artificial chromosomes (YACs) containing alphoid DNA from the centromere region of human chromosome 21 in a recombination-deficient yeast host. When these modified YACs were introduced into cultured human cells, a YAC with the alphoid DNA from the alpha21-I locus, containing CENP-B boxes at a high frequency and a regular repeat array, efficiently formed minichromosomes that were maintained stably in the absence of selection and bound CENP-A, CENP-B, CENP-C and CENP-E. The minichromosomes, 1-5 Mb in size and composed of multimers of the introduced YAC DNA, aligned at metaphase plates and segregated to opposite poles correctly in anaphase. Extensive cytological analyses strongly suggested that the minichromosomes had not acquired host sequences and were formed in all cases by a de novo mechanism. In contrast, minichromosomes were never produced with a modified YAC containing alphoid DNA from the alpha21-II locus, which contains no CENP-B boxes and has a less regular sequence arrangement. We conclude that alpha21-I alphoid DNA can induce de novo assembly of active centromere/kinetochore structures on minichromosomes.

Autoantigens↗
PubMed1998

Construction of YAC-based mammalian artificial chromosomes.

To construct a mammalian artificial chromosome (MAC), telomere repeats and selectable markers were introduced into a 100 kb yeast artificial chromosome (YAC) containing human centromeric DNA. This YAC, which has a regular repeat structure of alpha-satellite DNA and centromere protein B (CENP-B) boxes, efficiently formed MACs that segregated accurately and bound CENP-B, CENP-C, and CENP-E. The MACs appear to be about 1-5 Mb in size and contain YAC multimers. Structural analyses suggest that the MACs have not acquired host sequences and were formed by a de novo mechanism. The accurate segregation of the MACs suggests they have potential as vectors for introducing genes into mammals.

Animals↗
PubMed1998

Engineering mammalian chromosomes.

Construction of a mammalian artificial chromosome (MAC) will develop our understanding of the requirements for normal chromosome maintenance, replication and segregation while offering the capacity for introducing genes into cells. Construction of MACs with telomere, centromere and replication function has been approached by two methods. The 'top down' strategy uses artificially induced chromosome truncations as a means to define a minimal chromosome that retains the mitotic properties of a normal chromosome. The 'build up' approach has focused on attempts to assemble MAC vectors containing functionally defined telomere repeats together with candidate centromere and replication origin sequences. Here we report on significant advances in both areas, with particular emphasis on two reports showing that stable, low copy number MACs containing a functional centromere can be produced following transfection of naked DNA into the human HT1080 cell line. One approach used a transfection mixture of cloned synthetic alpha-satellite arrays up to 1 Mb in length and unlinked telomeric DNA, in either the presence or absence of random human genomic DNA fragments. In the second approach, MACs were formed from a defined yeast artificial chromosome (YAC) DNA molecule containing 100 kb of highly homo- geneous alphoid DNA retrofitted with human telomere repeats. These results demonstrate for the first time that alpha-satellite DNA can seed de novo centromeres in human cells, indicating that this repetitive sequence family plays an important role in centromere function. The stability of these MACs suggests that they have potential to be developed as gene delivery vectors.

Animals↗
PubMed1995

A hybrid sigma subunit directs RNA polymerase to a hybrid promoter in Escherichia coli.

Most of the sigma (transcriptional initiation specificity) subunits of RNA polymerase, from a wide range of eubacteria, show strong elements of amino acid sequence similarity. There is evidence that two of the "conserved" regions, 2.4 and 4.2, are involved in recognition of the consensus DNA sequences centred near -10 and -35, respectively, which define promoter sites for the initiation of transcription. Since all the alternative sigma subunits of the above type function by binding to a common core polymerase enzyme in a given bacterium, it can be predicted that a hybrid sigma might be functional, and if so should permit RNA polymerase to initiate only at a correspondingly hybrid promoter. To test these predictions, a hybrid gene encoding the amino-proximal 529 amino acids of the major Escherichia coli sigma protein, sigma 70 (including region 2.4) followed by the last 82 amino acids of the heat-shock sigma protein, sigma 32 (including region 4.2) was constructed and fused to Plac on a plasmid. Major-consensus, heat-shock and hybrid promoters were fused to a chloramphenicol acetyl transferase (CAT) reporter gene on a compatible plasmid. CAT assays showed that, as predicted, a promoter with a "heat-shock" -35 consensus and a "major" -10 consensus sequence (PHM) required Plac-dependent production of the hybrid sigma (sigma 70-32) for activity in vivo. PHM then became a strong promoter. The hybrid sigma gene has potential advantages over its parents for structure-function studies.

Amino Acid Sequence↗
PubMed1994

Role of the sigma 70 subunit of Escherichia coli RNA polymerase in transcription activation.

The role of the sigma 70 subunit of Escherichia coli RNA polymerase in transcription activation by positive transcription factors was investigated. For this purpose, we constructed a nested set of E. coli rpoD deletion mutants generating carboxy-terminally truncated sigma 70 subunits of RNA polymerase in a high-expression plasmid. The purified mutant sigma 70 subunits were reconstituted into holoenzymes and examined in vitro for their promoter selectivity. As expected, since the -35 recognition helix of sigma 70 was deleted in all cases, the mutant enzymes were unable to initiate at factor-independent promoters, except for the special case of perfect "extended minus 10" promoters, at which the need for -35 sequence recognition by RNA polymerase is replaced by recognition of additional base-pairs in the -10 region. However, two factor-dependent promoters, PhoB-dependent PpstS and cAMP receptor protein (CRP)-dependent P1gal, could be activated for transcription by different subsets of the mutant holoenzymes, although these promoters do not contain the perfect extended -10 sequences. These results establish that -35 DNA recognition by sigma 70 is not essential for these cases. Presumably it is replaced by protein-protein contacts between RNA polymerase and the activator, which in both cases is bound to the DNA in a position overlapping the -35 region. Further, the detailed results support the view that the contact and/or activation sites for these two factors may lie on the sigma 70 subunit, within a "contact site II", which extends at least from conserved region 3.2 to the upstream end of region 4.2. Moreover, as in the case of contact site I on the alpha subunit, it appears that contact site II contains various different subsites for interaction with specific class II activators, and that PhoB and CRP require distinct subsites.

Base Sequence↗