A modular instrument for exploring the mechanics of cardiac myocytes.

The cardiac ventricular myocyte is a key experimental system for exploring the mechanical properties of the diseased and healthy heart. Millions of primary myocytes, which remain viable for 4-6 h, can be readily isolated from animal models. However, currently available instrumentation allows the mechanical properties of only a few physically loaded myocytes to be explored within 4-6 h. Here we describe a modular and inexpensive prototype instrument that could form the basis of an array of devices for probing the mechanical properties of single mammalian myocytes in parallel. This device would greatly increase the throughput of scientific experimentation and could be applied as a high-content screening instrument in the pharmaceutical industry. The instrument module consists of two independently controlled Lorentz force actuators-force transducers in the form of 0.025 x 1 x 5 mm stainless steel cantilevers with 0.5 m/N compliance and 360-Hz resonant frequency. Optical position sensors focused on each cantilever provide position and force resolution of <1 nm/ radicalHz and <2 nN/ radicalHz, respectively. The motor structure can produce peak displacements and forces of +/-200 mum and +/-400 microN, respectively. Custom Visual Basic.Net software provides data acquisition, signal processing, and digital control of cantilever position. The functionality of the instrument was demonstrated by implementation of novel methodologies for loading and attaching healthy mammalian ventricular myocytes to the force sensor and actuator and use of stochastic system identification techniques to measure their passive dynamic stiffness at various sarcomere lengths.

The two Drosophila telomeric transposable elements have very different patterns of transcription.

The transposable elements HeT-A and TART constitute the telomeres of Drosophila chromosomes. Both are non-long terminal repeat (LTR) retrotransposons, sharing the remarkable property of transposing only to chromosome ends. In addition, strong sequence similarity of their gag proteins indicates that these coding regions share a common ancestor. These findings led to the assumption that HeT-A and TART are closely related. However, we now find that these elements produce quite different sets of transcripts. HeT-A produces only sense-strand transcripts of the full-length element, whereas TART produces both sense and antisense full-length RNAs, with antisense transcripts in more than 10-fold excess over sense RNA. In addition, features of TART sequence organization resemble those of a subclass of non-LTR elements characterized by unequal terminal repeats. Thus, the ancestral gag sequence appears to have become incorporated in two different types of elements, possibly with different functions in the telomere. HeT-A transcripts are found in both nuclear and cytoplasmic cell fractions, consistent with roles as both mRNA and transposition template. In contrast, both sense and antisense TART transcripts are almost entirely concentrated in nuclear fractions. Also, TART open reading frame 2 probes detect a cytoplasmic mRNA for reverse transcriptase (RT), with no similarity to TART sequence 5' or 3' of the RT coding region. This RNA could be a processed TART transcript or the product of a "free-standing" RT gene. Either origin would be novel. The distinctive transcription patterns of both HeT-A and TART are conserved in Drosophila yakuba, despite significant sequence divergence. The conservation argues that these sets of transcripts are important to the function(s) of HeT-A and TART.

Stability of tandem repeats in the Drosophila melanogaster Hsr-omega nuclear RNA.

The Drosophila melanogaster Hsr-omega locus produces a nuclear RNA containing > 5 kb of tandem repeat sequences. These repeats are unique to Hsr-omega and show concerted evolution similar to that seen with classical satellite DNAs. In D. melanogaster the monomer is approximately 280 bp. Sequences of 19 1/2 monomers differ by 8 +/- 5% (mean +/- SD), when all pairwise comparisons are considered. Differences are single nucleotide substitutions and 1-3 nucleotide deletions/insertions. Changes appear to be randomly distributed over the repeat unit. Outer repeats do not show the decrease in monomer homogeneity that might be expected if homogeneity is maintained by recombination. However, just outside the last complete repeat at each end, there are a few fragments of sequence similar to the monomer. The sequences in these flanking regions are not those predicted for sequences decaying in the absence of recombination. Instead, the fragmentation of the sequence homology suggests that flanking regions have undergone more severe disruptions, possibly during an insertion or amplification event. Hsr-omega alleles differing in the number of repeats are detected and appear to be stable over a few thousand generations; however, both increases and decreases in repeat numbers have been observed. The new alleles appear to be as stable as their predecessors. No alleles of less than approximately 5 kb nor more than approximately 16 kb of repeats were seen in any stocks examined. The evidence that there is a limit on the minimum number of repeats is consistent with the suggestion that these repeats are important in the function of the unusual Hsr-omega nuclear RNA.

Drosophila telomere transposon HeT-A produces a transcript with tightly bound protein.

Telomeres from Drosophila appear to be very different from those of other organisms. A transposable element, HeT-A, plays a major role in forming telomeres and may be the sole structural element, since telomerase-generated repeats are not found. The structure of the HeT-A element, deduced from cloned fragments of DNA, suggests that transposition of the element is mediated by a polyadenylylated RNA intermediate. We now report analyses of HeT-A transcripts. The major RNA is of the appropriate size and strandedness to serve as a transposition intermediate. This RNA is found in cultured cells and in intact flies and is unusual in that it is associated with protein after treatments that apparently remove all protein from other RNAs.

The nucleus-limited Hsr-omega-n transcript is a polyadenylated RNA with a regulated intranuclear turnover.

The Drosophila Hsr-omega puff, one of the largest heat shock puffs, reveals a very unusual gene, identified by heat shock but constitutively active in nearly all cell types. Surprisingly, Hsr-omega yields two transcription end-products with very different roles. The larger, omega-n, is a nuclear RNA with characteristics suggesting a new class of nuclear RNAs. Although it neither leaves the nucleus nor undergoes processing, omega-n RNA is polyadenylated, showing that polyadenylation is not limited to cytoplasmic RNA, but possibly has a function in the nucleus. The amount of omega-n within the nucleus is specifically regulated by both transcription and turnover. Heat shock and several other agents cause rapid increases in omega-n. A rapid return to constitutive levels follows withdrawal of the agents. Degradation of omega-n is inhibited by actinomycin D, suggesting a novel intranuclear mechanism for RNA turnover. Within the nucleus, some omega-n RNA is concentrated at the transcription site; however, most is evenly distributed over the nucleus, showing no evidence of a concentration gradient which might be produced by simple diffusion from the site of transcription. Previous studies suggested that omega-n has a novel regulatory role in the nucleus. The actinomycin D-sensitive degradation system makes possible rapid changes in the amount of omega-n, allowing the putative regulatory activities to reflect cellular conditions at a given time. Omega-n differs from the best studied nuclear RNAs, snRNAs, in many ways. Omega-n demonstrates the existence of intranuclear mechanisms for RNA turnover and localization that may be used by a new class of nuclear RNAs.

Hsr-omega, A Novel Gene Encoded by a Drosophila Heat Shock Puff.

Although originally identified because of its abundant transcription in heat shock, the hsr-omega gene is active, at generally lower levels, in non-stressed cells. The locus produces an unusual set of three transcripts. Evidence from a variety of experiments suggests that one of these transcripts acts in the nucleus, possibly to regulate the activity of a nuclear protein. Another of the transcripts appears to act in the cytoplasm, possibly monitoring or regulating some aspect of translation. The two transcripts together could have a role in coordinating nuclear and cytoplasmic activity. A number of processes occur in eukaryotic cells in which nuclear and cytoplasmic activities need to be coordinated; we suggest that hsr-omega plays a role in such coordination.