DNA methylation precedes chromatin modifications under the influence of the strain-specific modifier Ssm1

Ssm1 is responsible for the mouse strain-specific DNA methylation of the transgene HRD. In adult mice of the C57BL/6 (B6) strain, the transgene is methylated at essentially all CpGs. However, when the transgene is bred into the DBA/2 (D2) strain, it is almost completely unmethylated. Strain-specific methylation arises during differentiation of embryonic stem (ES) cells. Here we show that Ssm1 causes striking chromatin changes during the development of the early embryo in both strains. In undifferentiated ES cells of both strains, the transgene is in a chromatin state between active and inactive. These states are still observed 1 week after beginning ES cell differentiation. However, 4 weeks after initiating differentiation, in B6, the transgene has become heterochromatic, and in D2, the transgene has become euchromatic. HRD is always expressed in D2, but in B6, it is expressed only in early embryos. The transgene is already more methylated in B6 ES cells than in D2 ES cells and becomes increasingly methylated during development in B6, until essentially all CpGs in the critical guanosine phosphoribosyl transferase core are methylated. Clearly, DNA methylation of HRD precedes chromatin compaction and loss of expression, suggesting that the B6 form of Ssm1 interacts with DNA to cause strain-specific methylation that ultimately results in inactive chromatin.     

Studies on the genetics of tda-1 XY sex reversal in the mouse

When the Y chromosome of at least some populations of the house mouse of Western Europe and the Mediterranean, Mus musculus domesticus, is placed into the C57BL/6J (B6) inbred mouse genome, XY fetuses develop into hermaphrodites or females. It has been hypothesized that the testis-determining gene on the Y chromosome of M. m. domesticus (TdyDOM) interacts improperly with a putative B6/J recessive, testis-determining, autosomal gene (tda-1). The present study extended these earlier findings. The mating of B6 mice possessing the Y chromosome of M. m. domesticus (B6.YDom/Na; N6-N9) to females of the AKR, BALB/c, C3H/An, and C3H/He, but not SJL, strains resulted in aberrant testicular differentiation in day-14/15 F1 fetuses. The aberrant testes were characterized by a delay in testicular differentiation at the cranial and caudal poles of the gonad, i.e., the presence of a thin (or no) tunica albuginea and the presence of disorganized (or no) seminiferous tubules. Crossing B6.YDom male phenotypes with SJL females did not result in aberrant testicular differentiation, suggesting that the SJL strain possesses the dominant testis-determining, autosomal-1 allele, Tda-1. Studies using recombinant DNA probes specific for the murine Y chromosome have suggested that the SJL and AKR strains possess the M. m. domesticus Y chromosome. When Y chromosomes of the SJL and AKR strains were placed on the B6 background, aberrant testicular differentiation similar to tda-1 XY sex reversal occurred in only 1 out of 87 (1%) N4 day-14/15 fetuses possessing YSJL, but in 25 out of 45 (56%) N4 day-14/15 fetuses possessing YAKR.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of a region of genetic variability among Bacillus anthracis strains and related species.

The identification of a region of sequence variability among individual isolates of Bacillus anthracis as well as the two closely related species, Bacillus cereus and Bacillus mycoides, has made a sequence-based approach for the rapid differentiation among members of this group possible. We have identified this region of sequence divergence by comparison of arbitrarily primed (AP)-PCR "fingerprints" generated by an M13 bacteriophage-derived primer and sequencing the respective forms of the only polymorphic fragment observed. The 1,480-bp fragment derived from genomic DNA of the Sterne strain of B. anthracis contained four consecutive repeats of CAATATCAACAA. The same fragment from the Vollum strain was identical except that two of these repeats were deleted. The Ames strain of B. anthracis differed from the Sterne strain by a single-nucleotide deletion. More than 150 nucleotide differences separated B. cereus and B. mycoides from B. anthracis in pairwise comparisons. The nucleotide sequence of the variable fragment from each species contained one complete open reading frame (ORF) (designated vrrA, for variable region with repetitive sequence), encoding a potential 30-kDa protein located between the carboxy terminus of an upstream ORF (designated orf1) and the amino terminus of a downstream ORF (designated lytB). The sequence variation was primarily in vrrA, which was glutamine- and proline-rich (30% of total) and contained repetitive regions. A large proportion of the nucleotide substitutions between species were synonymous. vrrA has 35% identity with the microfilarial sheath protein shp2 of the parasitic worm Litomosoides carinii.     

Factors that control the differentiation of stem cells. I. The change in direction of hematopoietic stem cell differentiation under the effect of differentiating T-lymphocytes]

A mixed transplantation of bone marrow cells, and lymph nodes or thymic cells of mice CBA strain into lethally irradiated hybrid recipients (CBAXC57B1)F1 is accompanied with changes in the differentiation pattern from a mainly erythroid to a mainly granuloid way. Thymectomy of either donor of bone marrow cells or recipients, or both, destroys the stem cell differentiation in the direction of granulopoieseis. Intact syngeneic lymphocytes normalize differentiation of the stem cells, but in the presence of tissue antigens these provide for the stem cell differentiation mainly in the direction of granulopoiesis. The differentiation of stem haemopoietic cells is accomplished under the thymic and lymphocyte control. T-differentiating lymphocytes (Td) are the lymphocytes controlling the stem cell differentiation.     

Differentiation between B. pertussis and B. parapertussis according to their fatty acid composition

The fatty acid composition of 3 B. pertussis strains and 2 B. parapertussis strains grown on casein-carbon agar (CCA) with 8% of sheep blood added and without blood, as well as B. parapertussis strain grown on beef-extract agar (BEA) has been studied by gas chromatography. The fatty acid profiles characteristic of B. pertussis and B. parapertussis were greatly different, as B. parapertussis has a considerable amount of methylene-hexadenocanoic acid, while containing less hexadecenoic and octadecanoic acids and more tetradecanoic acid. The fatty acid composition of 2- to 5-day Bordetella cultures grown on CCA with and without blood has no essential differences. Differences in the content of various fatty acids in B. parapertussis grown on CCA and BEA had no essential influence on the fatty acid profile. The specificity of the fatty acid composition of B. pertussis and B. parapertussis allows to use this characteristics for their differentiation.     

Use of randomly cloned DNA fragments for identification of Bacteroides thetaiotaomicron.

Randomly cloned fragments of DNA from Bacteroides thetaiotaomicron were used as hybridization probes for differentiation of B. thetaiotaomicron from closely related Bacteroides species. HindIII digestion fragments of DNA from B. thetaiotaomicron (type strain) were inserted into plasmid pBR322 and labeled with [alpha-32P]dCTP by nick translation. These labeled plasmids were screened for hybridization to HindIII digests of chromosomal DNA from type strains of the following human colonic Bacteroides species: B. thetaiotaomicron, Bacteroides ovatus, reference strain 3452-A (formerly part of B. distasonis), Bacteroides uniformis, Bacteroides fragilis, Bacteroides vulgatus, Bacteroides distasonis, Bacteroides eggerthii, and reference strain B5-21 (formerly B. fragilis subsp. a). Two of the five cloned fragments hybridized only to DNA from B. thetaiotaomicron. Each of these two fragments hybridized to the same DNA restriction fragment in five strains of B. thetaiotaomicron other than the strain from which the DNA was cloned. One of the cloned fragments (pBT2) was further tested for specificity by determining its ability to hybridize to DNA from 65 additional strains of colonic Bacteroides.     

Genetic and Epigenetic Control of the Proliferation and Differentiation of Mouse Epidermal Melanocytes in Culture

Serum-free culture of epidermal cell suspensions from neonatal skin of mice of strain C57BL/10JHir (B10) showed that α-melanocyte-stimulating hormone (α-MSH) was involved in regulating the differentiation of melanocytes by inducing tyrosinase activity, melanosome formation, and dendritogenesis. Dibutyryl adenosine 3′:5′-cyclic monophosphate (DB-cAMP) similarly induced the differentiation of melanocytes. On the other hand, DBcAMP induced the proliferation of epidermal melanocytes in culture in the presence of keratinocytes. Basic fibroblast growth factor (bFGF) was also shown to stimulate the sustained proliferation of undifferentiated melanoblasts in the presence of DBcAMP and keratinocytes. These results suggest that the proliferation and differentiation of mouse epidermal melanoblasts and melanocytes in culture are regulated by the three factors; namely, cAMP, bFGF, and keratinocyte-derived factors. Moreover, serum-free primary culture of mouse epidermal melanocytes derived from B10 congenic mice, which carry various coat color genes, showed that the coat color genes were involved in regulating the proliferation and differentiation of mouse epidermal melanocytes by controlling the proliferative rate, melanosome formation and maturation, and melanosome distribution.     

Genetic and epigenetic control of the proliferation and differentiation of mouse epidermal melanocytes in culture.

Serum-free culture of epidermal cell suspensions from neonatal skin of mice of strain C57BL/10JHir (B10) showed that alpha-melanocyte-stimulating hormone (alpha-MSH) was involved in regulating the differentiation of melanocytes by inducing tyrosinase activity, melanosome formation, and dendritogenesis. Dibutyryl adenosine 3':5'-cyclic monophosphate (DBcAMP) similarly induced the differentiation of melanocytes. On the other hand, DBcAMP induced the proliferation of epidermal melanocytes in culture in the presence of keratinocytes. Basic fibroblast growth factor (bFGF) was also shown to stimulate the sustained proliferation of undifferentiated melanoblasts in the presence of DBcAMP and keratinocytes. These results suggest that the proliferation and differentiation of mouse epidermal melanoblasts and melanocytes in culture are regulated by the three factors; namely, cAMP, bFGF, and keratinocyte-derived factors. Moreover, serum-free primary culture of mouse epidermal melanocytes derived from B10 congenic mice, which carry various coat color genes, showed that the coat color genes were involved in regulating the proliferation and differentiation of mouse epidermal melanocytes by controlling the proliferative rate, melanosome formation and maturation, and melanosome distribution.     

Differentiation and melanin production in hyaline and pigmented strains of Microdochium bolleyi

The effect of various compounds on growth, melanin biosynthesis and cell differentiation was studied in a hyaline (SH25) and a pigmented (SH25B) strain of Microdochium bolleyi. Dark pigment production by the hyaline strain was induced by the presence of DOPA and indole in the medium, both of which are intermediates in the tyrosine pathway of melanin biosynthesis. These substrates also induced conidia and chlamydospore production, structures which were not normally seen in the hyaline strain. The systemic fungicide, tricyclazole, inhibited melanin synthesis in the pigmented strain but not the development of chlamydospore-like cell structures.     

Precise molecular weight determination of PCR products of the rRNA intergenic spacer region using electrospray quadrupole mass spectrometry for differentiation of B. subtilis and B. atrophaeus, closely related species of bacilli

Assessment of 16S–23S rRNA intergenic spacer region (ISR) sequence variability is an important supplement to 16S rRNA sequencing for differentiating closely related bacterial species. Species differentiation can also be achieved by determination of approximate size of PCR (polymerase chain reaction) products of ISRs, based on their relative electrophoretic mobility on agarose gels. Closely-related species can have ISR PCR products that are similar in size. More precise molecular weight (M.W.) determination of these products might allow improved discrimination of such species. Electrospray quadrupole mass spectrometry (ESI-Q-MS) has the potential to provide such precision. For ESI-Q-MS analysis, size limitation of PCR products is currently limited to around 130 base pairs (bp). Bacillus subtilis and Bacillus atrophaeus are two closely related species with few distinguishing phenotypic characteristics. B. subtilis has recently been sub-divided into two subgroups, W23 (type strain, W23) and 168 (type strain, 168). PCR products amplified from the ISR including the 5′ terminal end of the 23S rRNA and a conserved portion of the ISR were analyzed by ESI-Q-MS. A 119 or 120 bp PCR product was produced for B. atrophaeus strains. However, strains of B. subtilis subgroups W23 and 168 each produced 114 bp products. In summary, a mass spectrometry method was developed for differentiation of B. subtilis and B. atrophaeus. Also, the genetic similarity of B. subtilis subgroups W23 and 168 was confirmed. Accurate determination of the molecular weight of PCR products from the 16S–23S rRNA intergenic spacer region using electrospray quadrupole mass spectrometry has great potential as a general technique for characterizing closely related bacterial species.     

Calcium ions and the differentiation of Streptomyces hygroscopicus 155, a producer of an antibiotic complex]

The effect of Ca2+ on differentiation of Streptomyces hygroscopicus 155 and its inactive variant 155-0 was studied. Addition of Ca2+ to the medium induced formation of the aerial mycelium in the inactive variant and accelerated formation of the aerial mycelium in the parent strain. The inhibitory effect of EGTA, verapamil, nifedipin, chlorpromazine and dilthiazeme on the aerial mycelium formation demonstrated the physiological role of Ca2+ in the process. Addition of pandavir (nigericin) and azalomycin B, the antibiotics produced by the streptomycete, induced formation of the aerial mycelium in the inactive variant. The effect was higher in the presence of Ca2+. Streptomyces hygroscopicus 155 and its inactive variant synthesized a proteolytic complex containing metalloproteases and trypsin-like proteases. The total proteolytic activity of the inactive variant was lower than that of the parent strain. Addition of Ca2+ to the medium stimulated their proteolytic activity. The inducing action of the antibiotics produced by the parent strain on differentiation of S.hygroscopicus 155-0 and the increase of their action in the presence of Ca2+ suggested that they controlled the differentiation and that such a function of the antibiotics expressed itself through the Ca2+ signal system.     

Dictyopyrones, novel alpha-pyronoids isolated from Dictyostelium spp., promote stalk cell differentiation in Dictyostelium discoideum

Dictyopyrones A and B (DpnA and B), whose function(s) is not known, were isolated from fruiting bodies of Dictyostelium discoideum. In the present study, to assess their function(s), we examined the effects of Dpns on in vitro cell differentiation in D. discoideum monolayer cultures with cAMP. Dpns at 1-20 microM promoted stalk cell formation to some extent in the wild-type strain V12M2. Although Dpns by themselves could hardly induce stalk cell formation in a differentiation-inducing factor (DIF)-deficient strain HM44, both of them dose-dependently promoted DIF-1-dependent stalk cell formation in the strain. In the sporogenous strain HM18, Dpns at 1-20 microM suppressed spore formation and promoted stalk cell formation in a dose-dependent manner. Analogs of Dpns were less effective in affecting cell differentiation in both HM44 and HM18 cells, indicating that the activity of Dpns should be chemical structure specific. It was also shown that DpnA at 2-20 microM dose-dependently suppressed spore formation induced with 8-bromo cAMP and promoted stalk cell formation in V12M2 cells. Interestingly, it was shown by the use of RT-PCR that DpnA at 10 microM slightly promoted both prespore- and prestalk-specific gene expressions in an early phase of V12M2 and HM18 in vitro differentiation. The present results suggest that Dpns may have functions (1) to promote both prespore and prestalk cell differentiation in an early stage of development and (2) to suppress spore formation and promote stalk cell formation in a later stage of development in D. discoideum.     

Effect of low molecular weight regulators on the biosynthesis of rifamycin B by Amycolatopsis mediterranei strains

Formation of differentiation regulators of the A-factor group in representatives of Nocardia and Nocardia-like actinomyces: N. asteroides, N. brasiliensis, Amycolatopsis mediterranei and "Streptomyces listeri" was observed. The effect of the regulators of different nature (barbital, A-factor and B-factor) on biosynthesis of rifamycin B by A. mediterranei strains was studied. It was shown that the A-factor stimulated rifamycin B production in the adifferentiated low active variant isolated from a natural population of the active strain VNIIA 1713 of the rifamycin B-producing culture. B-Factor insignificantly inhibited biosynthesis of rifamycin B in the studied strains of A. mediterranei.     

Susceptibility of mice to phenytoin-induced cleft palate correlated with inhibition of fetal palatal RNA and protein synthesis (41255)

Phenytoin administered to pregnant mice during the critical embryonic period of palatal differentiation produced 50% cleft palates in the Ajax (A/J) strain compared to 1.6% clefts in the C57BL/6 (B6) strain of mice. Furthermore, a single maternal injection of phenytoin produced a significantly greater and more persistent decrease in fetal palatal RNA and protein synthesis in the sensitive A/J strain compared to that in the insensitive B6 strain of mice. Thus, these differential effects of phenytoin on RNA and protein synthesis are associated with the differential susceptibility to the teratogenic action of phenytoin in the two strains.     

Uniaxial mechanical strain modulates the differentiation of neural crest stem cells into smooth muscle lineage on micropatterned surfaces

Neural crest stem cells (NCSCs) play an important role in the development and represent a valuable cell source for tissue engineering. However, how mechanical factors in vivo regulate NCSC differentiation is not understood. Here NCSCs were derived from induced pluripotent stem cells and used as a model to determine whether vascular mechanical strain modulates the differentiation of NCSCs into smooth muscle (SM) lineage. NCSCs were cultured on micropatterned membranes to mimic the organization of smooth muscle cells (SMCs), and subjected to cyclic uniaxial strain. Mechanical strain enhanced NCSC proliferation and ERK2 phosphorylation. In addition, mechanical strain induced contractile marker calponin-1 within 2 days and slightly induced SM myosin within 5 days. On the other hand, mechanical strain suppressed the differentiation of NCSCs into Schwann cells. The induction of calponin-1 by mechanical strain was inhibited by neural induction medium but further enhanced by TGF-β. For NCSCs pre-treated with TGF-β, mechanical strain induced the gene expression of both calponin-1 and SM myosin. Our results demonstrated that mechanical strain regulates the differentiation of NCSCs in a manner dependent on biochemical factors and the differentiation stage of NCSCs. Understanding the mechanical regulation of NCSC differentiation will shed light on the development and remodeling of vascular tissues, and how transplanted NCSCs respond to mechanical factors.     

The use of multilocus sequence typing (MLST) and randomly amplified polymorphic DNA (RAPD) for the differentiation between strains of Burkholderia mallei

Burkholderia mallei is highly pathogenic microorganism for both humans and animals. In this work, the possibility of the use of the genotyping method for differentiation between strains of B. mallei was studied. A collection of 14 isolates of B. mallei was characterized using randomly amplified polymorphic DNA (RAPD) and multilocus sequence typing (MLST). RAPD was the best method used for detecting strain differences of B. mallei. It was suggested that this method would be an increasingly useful molecular epidemiological tool.