Defining ruminal and total-tract starch degradation for adult dairy cattle using in vivo data.

Literature was searched for studies performed in adult dairy cattle that simultaneously measured starch degradability in the rumen (RSDeg) and starch digestion in the total tract to compute postruminal starch digestion (PRSDig). Forty-one studies with 161 dietary treatments were used to form the data set. Of these diets, the major starch source was corn for 83 diets, small grain for 58 diets, and sorghum for 8 diets. Corn RSDeg was more variable than other sources. As measured in vivo across all starch sources, the percent RSDeg was influenced only by the amount of starch consumed, with the amount of degradation being approximately 75% at low starch intakes and decreasing to about 60% when 4 kg or more of starch were consumed. Small grain starch had greater RSDeg than corn or sorghum starch, which were approximately equal. The PRSDig of corn and small grain starches were approximately equal, but sorghum was about 15% less. Across all diets, models derived from the Cornell Net Carbohydrate Protein System predicted percentage of total-tract digestibility of starch very accurately, but overpredicted RSDeg and, as a result, underpredicted percent PRSDig. Calculation of RSDeg using a French model predicted the mean RSDeg with greater accuracy but less precisely. The relative differences in RSDeg percent among starch sources was correctly predicted by these models. A model using a revised rate of digestion as a way of combining effects of starch type and processing was developed, which predicted corn starch RSDeg and PRSDig with greater accuracy than nutrition models but only slightly better than using the mean observed degradation or the French calculation. Inaccuracies in prediction of RSDeg may be due mainly to processing effects and particle sizes, but these were not well reported in literature studies and were difficult to estimate. More accurate assessment of RSDeg and PRSDig will require better and more consistent reporting of grain processing. Based on this study, the French calculation is the most accurate of the models examined, although adjustments will be required to improve accuracy.

Mouse pseudouridine synthase 1: gene structure and alternative splicing of pre-mRNA.

Evidence for the alternative splicing of the message for mouse pseudouridine synthase 1 (mPus1p) was found when several expressed sequence tag clones were completely sequenced. The genomic DNA for the MPUS1 gene (6.9 kb) was cloned from a mouse genomic library; the gene contains seven exons, of which three are alternatively spliced. In addition, one of the internal exons (exon VI) is unusually large. RNase protection analysis confirmed that several alternatively spliced messages were present in mouse tissues and cells in culture. A Western blot of total cellular protein from mouse tissues and cultured cells was reacted with an antibody specific for mPus1p; at least three proteins were detected. One protein corresponds to the predicted molecular mass of mPus1p (44 kDa) and is the most abundant. The two other isoforms, one 2 kDa larger and one 7 kDa smaller than mPus1p, were differentially expressed. The cDNA species for the three isoforms were cloned into expression plasmids; the proteins were synthesized in vitro and tested for pseudouridine synthase activity. The two isoforms, one containing an insert of 18 amino acids in a region of the enzyme assumed to be critical for activity, and the other, which has a deletion of the protein coding potential of two exons, were both inactive on tRNA substrates that mPus1p modifies.

Pseudouridine synthase 3 from mouse modifies the anticodon loop of tRNA.

A cDNA encoding mouse pseudouridine synthase 3 (mPus3p) has been cloned. The predicted protein has 34% identity with yeast pseudouridine synthase 3 (Pus3), an enzyme known to form pseudouridine at positions 38 and 39 in yeast tRNA. The cDNA is 1.7 kb, and when used as a probe on a Northern blot of total RNA from mouse tissues or cells in culture, a band at 1.8 kb was observed. The open reading frame codes for a protein of 481 amino acids with a predicted molecular mass of 55 552 Da. When mPus3p was in vitro translated and used in reactions with tRNA substrates from both yeast and humans, uridines at position 39 were modified to pseudouridine. In a tRNA substrate with a uridine at position 38 (human tRNA(Leu)), there was very slight formation of pseudouridine at that position after incubation with mPus3p.

Cloning and characterization of a mammalian pseudouridine synthase.

This report describes the cloning and characterization of a pseudouridine (psi) synthase from mouse that we have named mouse pseudouridine synthase 1 (mpus1p). The cDNA is approximately 1.5 kb and when used as a probe on a Northern blot of mouse RNA from tissues and cultured cells, several bands were detected. The open reading frame is 393 amino acids and has 35% identity over its length with yeast psi synthase 1 (pus1p). The recombinant protein was expressed in Escherichia coli and the purified protein converted specific uridines to psi in a number of tRNA substrates. The positions modified in stoichiometric amounts in vitro were 27/28 in the anticodon stem and also positions 34 and 36 in the anticodon of an intron containing tRNA. A human cDNA was also cloned and the smaller open reading frame (348 amino acids) was 92% identical over its length with mpus1p but is shorter by 45 amino acids at the amino terminus. The expressed recombinant human protein has no activity on any of the tRNA substrates, most probably the result of the truncated open reading frame.

Mental retardation and learning disabilities: conceptual and applied issues.

The relationship between mental retardation and learning disabilities is clouded by conceptual issues and current practices in applied (i.e., educational and noneducational) settings. In this article, we initially discuss whether mental retardation can be considered a concomitant disability associated with learning disabilities or whether these two disabilities are mutually exclusive categories. Conceptual issues related to this question are then reviewed to provide a perspective for viewing these two traditional areas of exceptionality. Emerging areas of concern in term of definition, classification, etiology, and lifelong issues are addressed. Attention is then given to applied issues that have a direct effect on the lives of individuals with disabilities. Specific topics include educational curricula, instruction, inclusion, and adult services and supports.

A life skills approach to mathematics instruction: preparing students with learning disabilities for the real-life math demands of adulthood.

Current mathematics instruction does not address the day-to-day needs of many students with learning disabilities. Although the vast majority of students with learning disabilities are not college bound, much of mathematics instruction provides college preparation. Too often, classes in mathematics ignore the skills needed in home and community and on the job. The present article examines the ways in which general mathematics instruction, focused on daily living skills, can easily be integrated into the classrooms of students with learning disabilities.

Modification of human U4 RNA requires U6 RNA and multiple pseudouridine synthases.

Small nuclear RNAs (snRNA), cofactors in the splicing of pre-mRNA, are highly modified. In this report the modification of human U4 RNA was studied using cell extracts and in vitro synthesized, and therefore unmodified, U4 RNA. The formation of pseudouridine (Psi) at positions 4, 72 and 79 in U4 RNA was dependent on an RNA-containing cofactor, since the activities in the extracts were micrococcal nuclease (MN) sensitive. Extracts were fractionated on glycerol gradients and there was a broad peak of reconstitution activity centered at 14 S. Reconstitution was not due to additional enzymatic activity, since the peak fraction was MN sensitive. Oligodeoxynucleotide-mediated RNase H digestion of U6 RNA in the extracts inhibited formation of Psi in U4 RNA. From glycerol gradient analysis we determined that exogenously added U4 RNA that is associated with U6 RNA (sedimentation velocity 16 S) was significantly higher in Psi content than U4 RNA not associated with U6 RNA (8 S). Competitive inhibitors of Psi synthases, 5-fluorouridine-containing (5-FU) wild-type and mutant U4 RNAs, were used to investigate formation of Psi in U4 RNA. Deletions and point mutations in these 5-FU-containing U4 RNAs affected their ability to inhibit Psi synthase in vitro. With the aid of these potent inhibitors it was determined that at least two separate activities modify the uridines at these positions.

Metabolism of pre-messenger RNA splicing cofactors: modification of U6 RNA is dependent on its interaction with U4 RNA.

The requirements for the formation of pseudouridine (psi) in U4 and U6 RNAs, cofactors in the splicing of pre-messenger RNA, were investigated in vitro using HeLa nuclear (NE) and cytoplasmic (S100) extracts. Maximal psi formation for both RNAs was extract order-dependent. Maximal psi formation in U4 RNA required incubation in S100 followed by the addition of NE, paralleling the in vivo maturation pathway of U4 RNA. In contrast, maximal formation of psi in U6 RNA required incubation in NE followed by the addition of S100 extract. Since U6 RNA does not exit the nucleus in vivo the contribution of S100 was investigated. In experiments where the extracts were treated with micrococcal nuclease to digest endogenous snRNAs, the efficient formation of psi in U6 RNA was dependent on the presence of U4 RNA, but not in U5 RNA or tRNA. When mutant U4 RNAs that inhibit or strengthen the interaction between U4 RNA, and U6 RNA were substituted for wild-type U4 RNA, the results confirmed the need for the interaction between these two RNAs for psi formation in U6 RNA. U6 RNA isolated from glycerol gradients after incubation in extracts had four times as much psi when associated with U4 RNA.

Localization of modified nucleotides in Schizosaccharomyces pombe spliceosomal small nuclear RNAs: modified nucleotides are clustered in functionally important regions.

The specific and dynamic RNA:RNA interactions between pre-mRNA and small nuclear RNAs (snRNAs), especially U2, U5, and U6 snRNAs, form the catalytic core and are at the heart of the spliceosome formation. The functionally important regions in the snRNAs correspond to the highly modified regions in snRNAs from human, rat, and plant cells. To better understand the importance of the modifications of snRNAs, we identified and localized the modified nucleotides in the five spliceosomal snRNAs of Schizosaccharomyces pombe cells. Twenty-two modified nucleotides, including base methylations, 2'-O-methylations, and pseudouridines, were found in the five spliceosomal snRNAs. The conservation of modified nucleotides between human and S. pombe snRNAs is striking. In addition, most of the modified nucleotides are in or around positions that form hydrogen bonds with the pre-mRNA or with other snRNAs. The results are consistent with the suggestion that modified nucleotides are clustered around functionally important regions of the spliceosomal snRNAs. These data provide the basis for further functional studies on posttranscriptional modifications in spliceosomal snRNAs.

Transition and students with learning disabilities: creating sound futures.

This article initiates a double issue that addresses a traditionally absent or rare piece in the system puzzle of preparing individuals with learning disabilities to meet the challenges of adulthood. Most professional efforts have focused on academic preparation for the 25 years or so that learning disabilities have been recognized. The rest of the person's adult adjustment (self-determination, life skills and community living, vocational preparation and employment, etc.) is presented within the framework of vertical and horizontal transitions, to organize the reader to consider all the options that youth must consider and prepare for. Individualized transition planning is discussed as a dynamic vehicle by which to empower students and families to utilize strengths, set and reach short-term and long-range goals, and include community variables in the process. Finally, an overview of the two issues describes their creation, their core common themes, and highlights of each article.

Practical recommendations for using homework with students with learning disabilities.

Homework is a commonly used school practice. Its importance for students with learning disabilities has increased in recent years as these students have spent more instructional time in inclusive settings where homework is regularly given and as educational reforms have influenced the amount of homework being given. The first part of the article reviews selected issues that relate to using homework with students who have been identified as learning disabled. The major part of the article highlights effective (i.e., empirically validated) and recommended (i.e., suggested in the literature or determined by field-based reports) practices for using homework with this population.

Formation of pseudouridine in U5 small nuclear RNA.

The formation of pseudouridine (psi) on U5 small nuclear RNA (U5 snRNA) was studied using an in vitro modification system. Labeled U5 RNA, synthesized in vitro and therefore unmodified, was incubated in reactions containing S100 and/or nuclear extracts (NE) from HeLa cells, and the levels of psi were determined. There are three psi residues found in human U5 RNA, at positions 43, 46, and 53. Incubation of unmodified U5 RNA in reactions containing either S100 or NE supports psi formation at positions 43 and 46, which are found in a loop in the predicted secondary structure of U5 RNA. However, psi formation at position 53, which is found in a stem, is dependent on the presence of NE during the incubation. The order of extract addition does not have a significant effect on the formation of psi at position 53 as long as NE is present. The most efficient psi formation was observed with a combination of S100 and NE which allowed for efficient small nuclear ribonucleoprotein particle (snRNP) assembly and psi formation. When 9S and 20S U5 snRNPs were isolated by velocity sedimentation gradient centrifugation after incubation in the combined extracts, there was little difference in the psi levels at any of the positions for the two distinct particles. Mutations in the U5 RNA sequence do affect psi formation. U5 RNAs that have mutated Sm binding sites or are truncated prior to the Sm binding site have very low levels of psi formation at positions 43 and 46 and no detectable psi formation at position 53. A deletion of five nucleotides from 39 to 43 abolishes psi formation at positions 43 and 46, but the modification of position 53 is unaffected.

Pseudouridine formation in U2 small nuclear RNA.

U2 small nuclear RNA contains 13 pseudouridine (psi) nucleotides, of which 11 are clustered in 5' regions involved in base-pairing interactions with other RNAs in the spliceosome. As a first step toward understanding the psi formation pathway in U2 RNA, we investigated psi formation on unmodified human U2 RNA in a HeLa cell extract system. Psi formation was found to occur specifically within only those RNase T1 oligonucleotide fragments of U2 RNA known to contain psi in vivo. Using 5-fluorouridine (FUrd)-containing U2 RNAs as specific inhibitors of psi formation in non-FUrd-substituted substrate U2 RNA, we found that wild-type FUrd-containing U2 RNA as well as several FUrd-containing mutant U2 RNAs completely inhibited psi formation. In contrast, certain other mutant U2 RNAs containing FUrd displayed reduced inhibitory capacity. In these cases psi modifications occurred in specific RNase T1 fragments of the substrate U2 RNA only if the FUrd-containing competitor RNA was mutated at or near this site. Formation of psi at one site in U2 RNA appeared to be neither dependent on prior psi formation at another site or sites nor required for subsequent psi formation elsewhere in the molecule. This autonomous mode of psi formation may be driven by multiple psi synthase enzymes acting independently at different sites in U2 RNA.

Pseudouridine formation in small nuclear RNAs.

Recent in vitro studies on the formation of pseudouridine (psi) in the spliceosomal small nuclear RNAs (snRNA) are reviewed. Multiple psi synthase activities, in some cases more that one per snRNA, are responsible for this modification of uridine. There is a requirement for Sm protein binding for the efficient formation of psi in U5 RNA but not for the modification of U2 RNA. The inhibition of psi formation by the incorporation of 5-fluorouridine in the snRNA is also reviewed.

In vitro splicing of pre-messenger RNA with extracts from 5-fluorouridine-treated cells.

The effect of 5-fluorouridine (5-FU) treatment of cells on the splicing of pre-mRNA was determined using cellular extracts and splicing in vitro. Nuclear extracts from control cells and cells treated with 5-FU were prepared and used to splice pre-mRNAs in vitro. The drug treatment resulted in inhibition of cell growth but had little effect on RNA synthesis. The extracts from 5-FU-treated cells showed significant inhibition of splicing. This inhibition was the result of reduced efficiency and was not caused by a block at a specific step in the splicing pathway. There were no observable changes in the levels or physical properties of the small nuclear ribonucleoprotein particles that are essential cofactors in the splicing process. The deficiency in splicing in the extracts from 5-FU-treated cells could be supplemented by the addition of complementary fractions from a control extract.

Ribonucleoprotein particle assembly and modification of U2 small nuclear RNA containing 5-fluorouridine.

An in vitro assembly/modification system was used to study the effect of 5-fluorouridine (5-FU) incorporation on the biosynthesis of the U2 small nuclear ribonucleoprotein particle (U2 snRNP). Labeled U2 RNAs were transcribed in vitro with 5-fluoro-UTP either partially supplementing or completely replacing UTP during synthesis. The resulting U2 RNAs have levels of 5-fluorouridine that range from 0 to 100% of the uridine content. When incubated in reactions containing extracts from HeLa cells, these 5-FU U2 RNAs are assembled into RNPs that are recognized by anti-Sm monoclonal antibody even when there is a complete replacement of uridine with 5-FU. However, when the in vitro assembled U2 snRNPs are subjected to buoyant density gradient centrifugation, the particles that contain 100% 5-FU are not resistant to salt dissociation. When the in vitro assembled U2 snRNPs were analyzed by velocity sedimentation gradient centrifugation, 5-FU incorporation correlated with a shift in the sedimentation rate of the particles. With 100% 5-FU incorporation, the peak of radioactivity shifted to approximately 15 S (control U2 RNA was at approximately 12 S). This peak from 5-FU U2 snRNPs was not resistant to dissociation on cesium sulfate gradients. The amount of pseudouridine (psi) found in the RNA from snRNP assembled in vitro on control and 5-FU-containing U2 RNAs was determined, and even at very low levels of 5-FU incorporation (5% replacement), the formation of psi was severely inhibited (36% of control). At higher levels of 5-FU incorporation, there was essentially no psi formed.

Multiple pseudouridine synthase activities for small nuclear RNAs.

The formation of pseudouridine (psi) in human U1, U2 and U5 small nuclear RNAs (snRNAs) was investigated using HeLa cell extracts. Unmodified snRNAs were synthesized in vitro and the extent of psi formation was determined after incubation in cell extracts. The formation of psi on labelled substrates was monitored in the presence of 5-fluorouracil (5-FU)-containing snRNAs as inhibitors of psi formation. The conversion of uridine to psi was inhibited only when the cognate 5-FU-containing inhibitor snRNA was included in the reaction. For example, 5-FU-containing U1 RNA inhibited psi formation in unmodified U1 RNA, but not in (unmodified) U2 or U5 RNAs. The results suggest that there are at least three activities that form psi in these snRNAs. The 5-FU-containing RNAs were stable during incubation in the cell extracts. A 12-fold molar excess of unlabelled U1 RNA did not inhibit psi formation on a labelled U1 RNA substrate, whereas a 3-fold molar excess of 5-FU-containing U1 RNA nearly abolished psi formation on the U1 substrate. The fact that 5-FU-containing snRNAs are potent inhibitors of psi formation in these pre-mRNA splicing cofactors raises the possibility that this is related to the cytotoxicity of fluoropyrimidines in cancer chemotherapy.

Pseudouridine modification of U5 RNA in ribonucleoprotein particles assembled in vitro.

The formation of pseudouridine (psi) in U5 RNA during ribonucleoprotein (RNP) assembly was investigated by using HeLa cell extracts. In vitro transcribed, unmodified U5 RNA assembled into an RNP particle with the same buoyant density and sedimentation velocity as did U5 small nuclear RNP from extracts. The greatest amount of psi modification was detected when a combination of S100 and nuclear extracts was used for assembly. psi formation was inhibited when ATP and creatine phosphate or MgCl2 were not included in the assembly reaction, paralleling the inhibition of RNP particle formation. A time course of assembly and psi formation showed that psi modification lags behind RNP assembly and that at very early time points, Sm-reactive U5 small nuclear RNPs are not modified. Two of three psi modifications normally found in U5 RNA were present in RNA incubated in the extracts. Mutations in the form of deletions and truncations were made in the U5 sequence, and the effect of these mutations on psi formation was investigated. A mutation in the area of stem-loop I which contains the psi moieties or in the Sm binding sequence affected psi formation.