Histone H2B ubiquitylation disrupts local and higher-order chromatin compaction

Regulation of chromatin structure involves histone posttranslational modifications that can modulate intrinsic properties of the chromatin fiber to change the chromatin state. We used chemically defined nucleosome arrays to demonstrate that H2B ubiquitylation (uH2B), a modification associated with transcription, interferes with chromatin compaction and leads to an open and biochemically accessible fiber conformation. Notably, these effects were specific for ubiquitin, as compaction of chromatin modified with a similar ubiquitin-sized protein, Hub1, was only weakly affected. Applying a fluorescence-based method, we found that uH2B acts through a mechanism distinct from H4 tail acetylation, a modification known to disrupt chromatin folding. Finally, incorporation of both uH2B and acetylated H4 resulted in synergistic inhibition of higher-order chromatin structure formation, possibly a result of their distinct modes of action

Investigations of the Mechanism and Substrate Scope of the Enzymes Involved in Lantibiotic Biosynthesis

160 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2005.The subtilin prepeptide SpaS consists of 56 amino acids that includes five cysteine residues. This peptide is the substrate of the putative lantibiotic dehydratase, SpaB, and cyclase SpaC. The heterologously expressed and purified SpaS peptide was employed in assays with the purified SpaB and SpaC proteins to investigate conditions for the in vitro reconstitution of these proteins. The successful in vitro reconstitution of the lacticin 481 synthetase, LctM, led to preliminary investigations of its low substrate specificity. In this thesis the substrate specificity of LctM was tested further with non-proteinogenic amino acids. The effect of D-amino acids in the truncated prepeptide, LctA, on LctM action was determined. The ability of LctM to catalyze the cyclization of unusual substrates bearing cysteine analogues such as L-homocysteine was demonstrated. The mechanism of dehydration of Ser/Thr residues by LctM was also interrogated. Truncated substrates containing phosphorylated serine and threonine residues underwent dehydration by Lctm in the absence of ATP when ADP was present in the assays. In conjunction with the identification of phosphorylation in the structural region of two different truncated mutants, these results suggest that LctM may catalyze phosphorylation of Ser/Thr residues in its substrate prior to their dehydration. Mutations of highly conserved residues in the leader sequence as well as deletions of the N-terminal residues in the leader sequence of LctA did not affect dehydration by LctM.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD

Investigations of the Mechanism and Substrate Scope of the Enzymes Involved in Lantibiotic Biosynthesis

160 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2005.The subtilin prepeptide SpaS consists of 56 amino acids that includes five cysteine residues. This peptide is the substrate of the putative lantibiotic dehydratase, SpaB, and cyclase SpaC. The heterologously expressed and purified SpaS peptide was employed in assays with the purified SpaB and SpaC proteins to investigate conditions for the in vitro reconstitution of these proteins. The successful in vitro reconstitution of the lacticin 481 synthetase, LctM, led to preliminary investigations of its low substrate specificity. In this thesis the substrate specificity of LctM was tested further with non-proteinogenic amino acids. The effect of D-amino acids in the truncated prepeptide, LctA, on LctM action was determined. The ability of LctM to catalyze the cyclization of unusual substrates bearing cysteine analogues such as L-homocysteine was demonstrated. The mechanism of dehydration of Ser/Thr residues by LctM was also interrogated. Truncated substrates containing phosphorylated serine and threonine residues underwent dehydration by Lctm in the absence of ATP when ADP was present in the assays. In conjunction with the identification of phosphorylation in the structural region of two different truncated mutants, these results suggest that LctM may catalyze phosphorylation of Ser/Thr residues in its substrate prior to their dehydration. Mutations of highly conserved residues in the leader sequence as well as deletions of the N-terminal residues in the leader sequence of LctA did not affect dehydration by LctM.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD

Disulfide-directed histone ubiquitylation reveals plasticity in hDot1L activation

We have developed a readily accessible disulfide-directed methodology for the site-specific modification of histones by ubiquitin and ubiquitin-like proteins. The disulfide-linked analog of mono-ubiquitylated H2B stimulated the H3K79 methyltransferase activity of hDot1L to a similar extent as the native isopeptide linkage. This permitted structure-activity studies of ubiquitylated mononucleosomes that revealed plasticity in the mechanism of hDot1L stimulation and identified surfaces of ubiquitin important for activation

Not Available

Not AvailableExperiment was conducted on Jersey crossbred cows (12) to compare 2 management practices viz. hand and machine milking. Field investigations were also carried out on other milking management practices, IMI, hygiene status and cleanliness in 3 stages. A total of 459 milk samples were analysed. Farm experimentation revealed that SCC and MCMT were significantly higher in hand milking as compared to machine milking management. But milk yield and milk extraction rate were significantly lower in hand milking as compared to machine milking management. The time required for milking/animal was significantly higher in hand milking as compared to machine milking management. The morning and evening milk samples of both hand and machine milking showed higher values of SCC, MCMT, pH during evening as compared to morning session but milk yield and milk extraction rate were lower during evening as compared to morning session whereas time required for milking/animal was higher in morning as compared to evening time. Almost similar trend of fat and SNF (%) were estimated in both milking session. Field investigation indicated that SCC, MCMT, pH were higher in IMI animal as compared to no-IMI animal whereas fat and SNF were lower in IMI animal as compared to no IMI animal. Teat dipping and screening of udders for mastitis were never followed by any farmer. Most of farmer having single cow, maintained good hygiene status and cleanliness but most of the farmer having >3 cows, maintained poor hygiene status. Farm experimentation concluded that the IMI can be reduced in machine milking practices in comparison to hand milking practices with higher milk quantity without affecting milk composition in Jersey crossbred cows. Field investigation concluded that there is a significant association between animal keeping pattern and hygiene status/cleanliness at study area. So efforts should be made to increase cleanliness and hygiene status in milking cows itself, housing of animal and milkers of farmer’s house to reduce incidence of IMI in changing scenario

Histone H2B ubiquitylation disrupts local and higher-order chromatin compaction

Regulation of chromatin structure involves histone posttranslational modifications that can modulate intrinsic properties of the chromatin fiber to change the chromatin state. We used chemically defined nucleosome arrays to demonstrate that H2B ubiquitylation (uH2B), a modification associated with transcription, interferes with chromatin compaction and leads to an open and biochemically accessible fiber conformation. Notably, these effects were specific for ubiquitin, as compaction of chromatin modified with a similar ubiquitin-sized protein, Hub1, was only weakly affected. Applying a fluorescence-based method, we found that uH2B acts through a mechanism distinct from H4 tail acetylation, a modification known to disrupt chromatin folding. Finally, incorporation of both uH2B and acetylated H4 resulted in synergistic inhibition of higher-order chromatin structure formation, possibly a result of their distinct modes of action

Fluorescent Probes Reveal a Minimal Ligase Recognition Motif in the Prokaryotic Ubiquitin-like Protein from Mycobacterium tuberculosis

The prokaryotic ubiquitin-like protein (Pup)-based proteasomal system in the pathogen Mycobacterium tuberculosis (<i>Mtb</i>) is essential for its survival in a mammalian host. The Pup ligase enzyme, PafA, conjugates Pup to a suite of proteins targeted for proteasomal degradation and is necessary for persistent infection by <i>Mtb</i>. We report the design and application of fluorescent probes for use in elucidating the mechanisms of Pup and substrate recognition by PafA. Our studies revealed that the C-terminal 26 amino acid sequence of Pup is the minimal ligase recognition motif in <i>Mtb</i>. Specific hydrophobic residues within this sequence that are known to be important for the interactions of Pup with proteasomes are also critical for the activation of Pup by PafA

Influence of pre and postpartum alpha-tocopherol supplementation on milk yield, milk quality, and udder health of Jersey crossbred cows at tropical lower Gangetic region

Background and Aim: Alpha-tocopherol supplementation influences milk yield, milk quality, and udder health of dairy cows, which needs to be investigated for Jersey crossbred cows at hot-humid climate. Therefore, the present study was framed with an objective to study the effect of pre and postpartum Alpha-tocopherol supplementation on milk yield, milk quality, and udder health status of Jersey crossbred cows at tropical climate. Materials and Methods: For this study, 19 similar parity, body condition score, and production level dairy animals were separated randomly into three groups, namely, Control, T1 and T2. Control group (no supplementation) was compared with two treatment groups, namely, T1 and T2. Supplementation of Alpha-tocopherol was done in concentrate fed to the animals (at 1 g/cow/day) 30 days prepartum to 30 days postpartum in T1 and 30 days prepartum to 60 days postpartum in T2 groups. Observations were taken for different parameters up to 5 months of lactation. Results: Statistically analyzed data revealed that overall significantly (p0.05). Conclusion: Supplementation of Alpha-tocopherol during prepartum to initial lactation period may enhance milk yield, milk quality, and udder health status of Jersey crossbred cows at the tropical lower Gangetic region