In vitro preselection of gene-trapped embryonic stem cell clones for characterizing novel developmentally regulated genes in the mouse.

We have developed an in vitro gene trap screen for novel murine genes that allows one to determine, prior to making chimeric or transgenic animals, if these genes are expressed in one or more specific embryonic tissues. Totipotent embryonic stem (ES) cells are infected with a retroviral gene trap construct encoding a selectable lacZ/neo fusion gene, which is expressed only if the gene trap inserts within an active transcription unit. G418-resistant ES cell clones are induced to differentiate in vitro, and neurons, glia, myocytes, and chondrocytes are screened for expression of beta-galactosidase (beta-gal). cDNAs of the gene trap transcripts are obtained by 5' rapid amplification of cDNA ends and are sequenced to determine if they represent novel genes. In situ hybridization analyses show that trapped genes are expressed in vivo within the cell types that express beta-gal in vitro. Gene traps and their wild-type alleles are characterized in terms of copy number, alternate splicing of their transcripts, and the proportion of endogenous mRNA sequence that is replaced by lacZ/neo in the hybrid gene trap transcript. This approach, which we term "in vitro preselection," is more economical than standard in vivo gene trap screening because tissue-specific expression of probable knockout alleles is verified before transgenic animals are generated. These results also highlight the utility of ES cell differentiation in vitro as a method with which to study the molecular mechanisms regulating the specification and commitment of a variety of cell and tissue types.

Intramolecular base pairing between the nematode spliced leader and its 5' splice site is not essential for trans-splicing in vitro.

The spliced leader RNAs of both trypanosomes and nematodes can form similar secondary structures where the trans-splice donor site is involved in intramolecular base pairing with the spliced leader sequence. It has been proposed that this base pairing could serve to activate autonomously the SL RNA splice donor site. Here, we have examined exon requirements for trans-splicing in a nematode cell free system. Complete disruption of secondary structure interactions at and around the trans-splice donor site did not affect the ability of the SL RNA to function in trans-splicing. In addition, the highly conserved 22 nt sequence could be productively replaced by artificial exons ranging in size from 2 to 246 nucleotides. These results reinforce the view that the 'intron' portion of the SL RNA functions as an independent Sm snRNP whose role is to deliver exon sequences to the trans-spliceosome.

Structural measures as predictors of injury basketball players.

The purpose of this study was to investigate the relationship between structure and injury in basketball players, with the goal of developing equations to predict injury. We examined 45 subjects who were participating in a community center basketball league. Each subject was measured prior to the season for bilateral weight, quadricep girth, calf girth, Q-angle of the knee, dorsiflexion of the ankle, forefoot varus, rearfoot valgus, and true and apparent leg length. All lower extremity injuries causing a game to be missed were recorded during the 16-game season. Average values for bilateral weight, quadricep girth, Q-angle, rearfoot valgus, and leg length measures for the injured players were all larger than the average for the non-injured players by at least one standard deviation. A logistic regression equation using three of the variables correctly predicted the injury status of 91% of the players. The three-variable equation was then used prospectively to predict injury status for members of a small college basketball team. Only one player missed a game due to injury, and the equation identified that player as the most likely to be injured. This study demonstrates a strong relationship between structural measures and lower extremity injury in basketball players.

Transcription of a nematode trans-spliced leader RNA requires internal elements for both initiation and 3' end-formation.

We have used block substitution mutagenesis and in vitro transcription to define sequence elements important for efficient initiation and 3' end-formation of the trans-spliced leader RNA (SL RNA) of the parasitic nematode Ascaris lumbricoides. These experiments indicate that the SL RNA has an unusual promoter structure containing elements which include the 22 nt trans-spliced leader exon itself. Efficient transcription is correlated with the binding of a factor to the 22 nt (SL) sequence; mutations within the SL which abolish transcription lead to a loss in binding of this factor. In addition to internal sequences, synthesis of SL RNA in vitro requires an element centered 50 bases upstream of the cap site. Mutations within this element dramatically affect the level of SL RNA synthesis but do not affect accuracy of initiation. Finally, all of the information required for accurate 3' end-formation of SL RNA lies within the transcribed region. Thus, the arrangement of sequences necessary for the synthesis of SL RNAs does not resemble that of sequences important for the synthesis of vertebrate U snRNAs despite the similarities between SL RNAs and U snRNAs.

Characterization and expression of a spliced leader RNA in the parasitic nematode Ascaris lumbricoides var. suum.

The parasitic nematode Ascaris spp. contains a 22-nucleotide spliced-leader (SL) sequence identical to the trans-SL previously described in Caenorhabditis elegans and other nematodes. The SL comprises the first 22 nucleotides of a approximately 110-base RNA and is transcribed by RNA polymerase II. The SL RNA contains a trimethylguanosine cap and a consensus Sm binding site. Furthermore, the Ascaris SL RNA has the potential to adopt a secondary structure which is nearly identical to potential secondary structures of similar SL RNAs in C. elegans and Brugia malayi.

Analysis of glycosaminoglycans during chondrogenesis of normal and brachypod mouse limb mesenchyme.

Studies have been carried out to characterize radioactive incorporation rates and steady-state levels of hyaluronic acid (HA) and chondroitin sulfate (CS) as well as hyaluronidase activity in the hind limbs of normal and brachpod mouse embryos between 11-14 days of gestation. The results of the analysis show that changes in the synthetic and degradative rates of HA and CS occur at about the 12.5-day stage in normal hind limbs. These changes include an increased rate of CS synthesis, a decreased rate of HA synthesis, and a correspondingly sharp, transitory rise in hyaluronidase activity. Similar changes also occur in brachypod hind limbs but appear to be delayed in onset by approximatly one-half day. In addition, the mutant hind limb exhibits a slower loss of HA at a time when turnover would be expected to be on the increase. These changes are concomitant with cell surface alterations and abnormal mesenchymal condensation formation which have been previously shown to occur in this mutant.