A set of PCR primers was designed and validated for specific detection and quantification of Prevotella ruminicola, Prevotella albensis, Prevotella bryantii, Fibrobacter succinogenes, Selenomonas ruminantium-Mitsuokella multiacida, Streptococcus bovis, Ruminococcus flavefaciens, Ruminobacter amylophilus, Eubacterium ruminantium, Treponema bryantii, Succinivibrio dextrinosolvens, and Anaerovibrio lipolytica. By using these primers and the real-time PCR technique, the corresponding species in the rumens of cows for which the diet was switched from hay to grain were quantitatively monitored. The dynamics of two fibrolytic bacteria, F. succinogenes and R. flavefaciens, were in agreement with those of earlier, culture-based experiments. The quantity of F. succinogenes DNA, predominant in animals on the hay diet, fell 20-fold on the third day of the switch to a grain diet and further declined on day 28, with a 57-fold reduction in DNA. The R. flavefaciens DNA concentration on day 3 declined to approximately 10% of its initial value in animals on the hay diet and remained at this level on day 28. During the transition period (day 3), the quantities of two ruminal prevotella DNAs increased considerably: that of P. ruminicola increased 7-fold and that of P. bryantii increased 263-fold. On day 28, the quantity of P. ruminicola DNA decreased 3-fold, while P. bryantii DNA was still elevated 10-fold in comparison with the level found in animals on the initial hay diet. The DNA specific for another xylanolytic bacterium, E. ruminantium, dropped 14-fold during the diet switch and was maintained at this level on day 28. The concentration of a rumen spirochete, T. bryantii, decreased less profoundly and stabilized with a sevenfold decline by day 28. The variations in A. lipolytica DNA were not statistically significant. After an initial slight increase in S. dextrinosolvens DNA on day 3, this DNA was not detected at the end of the experiment. S. bovis DNA displayed a 67-fold increase during the transition period on day 3. However, on day 28, it actually declined in comparison with the level in animals on the hay ration. The amount of S. ruminantium-M. multiacida DNA also increased eightfold following the diet switch, but stabilized with only a twofold increase on day 28. The real-time PCR technique also uncovered differential amplification of rumen bacterial templates with the set of universal bacterial primers. This observation may explain why some predominant rumen bacteria have not been detected in PCR-generated 16S ribosomal DNA libraries.The complex symbiotic microbiota of the rumen is responsible for breakdown of plant fiber, an ability the ruminal host animals lack. This microbiota is highly responsive to changes in diet, age, antibiotic use, and the health of the host animal and varies according to geographical location, season, and feeding regimen (reviewed in references 4, 12, 21, and 31). In the early days of rumen microbiology, these changes were monitored by cultivation-based techniques, but limitations inherent in ...

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Molecular diversity of rumen archaea was analyzed by PCR amplification and sequencing of two 16S rRNA clone libraries prepared from the bovine rumen fluid using two different archaea-specific primer sets. The first library of 19 clones which was generated with primers D30 and D33, produced essentially two groups of sequences, one affiliated with Methanomicrobium mobile (21% of clones) and the other -- with the uncultured archaeal sequences from anaerobic digester, which are distantly associated with Thermoplasma (79% of clones). The second library of 25 clones, which was generated with primers 0025e Forward and 1492 Reverse, produced a higher degree of diversity: in addition to the previous two groups, with the M. mobile- (56%) and Thermoplasma-associated sequences (20%), four clones (16%) were identified as Methanobrevibacter spp. The remaining two sequences were associated with unidentified archaeal sequences from the rumen and swine waste. Phylogenetic placement of eight almost complete 16S rRNA sequences revealed the existence of a novel cluster of the rumen Euryarchaeota, which is not affiliated with the known methanogenic archaea.

Summary Hox proteins form complexes with Pbx and Meis cofactors to control gene expression, but the role of Meis is unclear. We demonstrate that Hoxb1-regulated promoters are highly acetylated on histone H4 (AcH4) and occupied by Hoxb1, Pbx and Meis in zebrafish tissues where these promoters are active. Inhibition of Meis blocks gene expression and reduces AcH4 levels at these promoters, suggesting a role for Meis in maintaining AcH4. Within Hox transcription complexes, Meis binds directly to Pbx and we find that this binding displaces histone deacetylases (HDACs) from Hoxb1-regulated promoters in zebrafish embryos. Accordingly, Pbx mutants that cannot bind Meis act as repressors by recruiting HDACs and reducing AcH4 levels, while Pbx mutants that bind neither HDAC nor Meis are constitutively active and recruit CBP to increase AcH4 levels. We conclude that Meis acts, at least in part, by controlling access of HDAC and CBP to Hox-regulated promoters.

Serum response factor and the (CC(A/T) 6 GG) (CArG) box interact to promote the transcription of c-fos and muscle genes; this tissue-specific activity may require co-regulators for serum response factor. The ␣ 1 integrin promoter contains two cis-elements besides the CArG box: a TAAT sequence, a consensus binding site for homeoproteins, and a GATA-binding box. As a candidate TAAT-binding factor, we cloned an NK family homeobox gene, Nkx-3.2, which is expressed mainly in smooth muscle tissues and skeletal structures. Nkx-3.2, serum response factor, and GATA-6 were co-expressed only in the medial smooth muscle layer of arteries. These three transcription factors formed a complex with their corresponding cis-elements and cooperatively transactivated smooth muscle genes, including ␣ 1 integrin, SM22␣, and caldesmon. Cardiac muscle-specific members of the NK and GATA families exist, and the triad of Nkx-2.5, serum response factor, and GATA-4 also transactivated the cardiac atrial natriuretic factor gene, which contains a CArG-like box, a GATA-binding box, and an NK-binding element. Our findings demonstrate that smooth and cardiac muscle have a shared transcriptional machinery and that the GATA and NK families confer muscle specificity on the serum response factor/CArG interaction.