Cloning of the Xenopus laevis aldolase C gene and analysis of its promoter function in developing Xenopus embryos and A6 cells

A Xenopus aldolase C gene (XAClambda3-1), much longer (9.6 kb) than human and rat genes (3.7-3.6 kb), was isolated and characterized, and expression studies were performed using Xenopus embryos and A6 cells, a kidney cell line constitutively expressing aldolase C gene. The Xenopus gene contained nine exons, and in its proximal 5'-upstream region a GC box and a 16 bp long aldolase C-specific element (ACSE), and in addition, a CCAAT box and a TATA-like element, both missing in mammalian genes. The lacZ gene connected to the 5'-upstream region (1.6 kb) of the aldolase gene containing many potentially regulative sequence elements was expressed in embryos temporally and spatially like the endogenous aldolase C gene. Deletion experiments using embryos and A6 cells suggested that this 5'-upstream DNA contained in its distal part a region which negatively affected on its expression in embryos, but not in A6 cells. The proximal-most region contained a basal promoter (68 bp) essential for expression in both embryos and A6 cells. Deletion experiments using A6 cells failed to detect such regulative regions within the first intron (spanning ca. 4 kb). Analyses with mutated promoters in A6 cells revealed that the GC box was the crucial element in the basal promoter, although the TATA-like element appeared to have a slightly stimulative effect on the GC box functioning. Gel retardation and foot-printing assays revealed the occurrence in A6 cells of a nuclear factor(s) that binds specifically to the GC box. Since Xenopus aldolase C gene has several unique structural features, we expect that it will provide an interesting material for studying the evolution and developmental control of the aldolase C gene.     

Distinct regulatory elements control muscle-specific, fiber-type-selective, and axially graded expression of a myosin light-chain gene in transgenic mice

The fast alkali myosin light chain 1f/3f (MLC1f/3f) gene is developmentally regulated, muscle specific, and preferentially expressed in fast-twitch fibers. A transgene containing an MLC1f promoter plus a downstream enhancer replicates this pattern of expression in transgenic mice. Unexpectedly, this transgene is also expressed in a striking (approximately 100-fold) rostrocaudal gradient in axial muscles (reviewed by J. R. Sanes, M. J. Donoghue, M. C. Wallace, and J. P. Merlie, Cold Spring Harbor Symp. Quant. Biol. 57:451-460, 1992). Here, we analyzed the expression of mutated transgenes to map sites necessary for muscle-specific, fiber-type-selective, and axially graded expression. We show that two E boxes (myogenic factor binding sites), a homeodomain (hox) protein binding site, and an MEF2 site, which are clustered in an approximately 170-bp core enhancer, are all necessary for maximal transgene activity in muscle but not for fiber-type- or position-dependent expression. A distinct region within the core enhancer promotes selective expression of the transgene in fast-twitch muscles. Sequences that flank the core enhancer are also necessary for high-level activity in transgenic mice but have little influence on activity in transfected cells, suggesting the presence of regions resembling matrix attachment sites. Truncations of the MLC1f promoter affected position-dependent expression of the transgene, revealing distinct regions that repress transgene activity in neck muscles and promote differential expression among intercostal muscles. Thus, the whole-body gradient of expression displayed by the complete transgene may reflect the integrated activities of discrete elements that regulate expression in subsets of muscles. Finally, we show that transgene activity is not significantly affected by deletion or overexpression of the myoD gene, suggesting that intermuscular differences in myogenic factor levels do not affect patterns of transgene expression. Together, our results provide evidence for at least nine distinct sites that exert major effects on the levels and patterns of MLC1f expression in adult muscles.     

Inefficient muscular stabilization of the lumbar spine associated with low back pain. A motor control evaluation of transversus abdominis

STUDY DESIGN: The contribution of transversus abdominis to spinal stabilization was evaluated indirectly in people with and without low back pain using an experimental model identifying the coordination of trunk muscles in response to a disturbances to the spine produced by arm movement. OBJECTIVES: To evaluate the temporal sequence of trunk muscle activity associated with arm movement, and to determine if dysfunction of this parameter was present in patients with low back pain. SUMMARY OF BACKGROUND DATA: Few studies have evaluated the motor control of trunk muscles or the potential for dysfunction of this system in patients with low back pain. Evaluation of the response of trunk muscles to limb movement provides a suitable model to evaluate this system. Recent evidence indicates that this evaluation should include transversus abdominis. METHODS: While standing, 15 patients with low back pain and 15 matched control subjects performed rapid shoulder flexion, abduction, and extension in response to a visual stimulus. Electromyographic activity of the abdominal muscles, lumbar multifidus, and the surface electrodes. RESULTS: Movement in each direction resulted in contraction of trunk muscles before or shortly after the deltoid in control subjects. The transversus abdominis was invariably the first muscle active and was not influenced by movement direction, supporting the hypothesized role of this muscle in spinal stiffness generation. Contraction of transversus abdominis was significantly delayed in patients with low back pain with all movements. Isolated differences were noted in the other muscles. CONCLUSIONS: The delayed onset of contraction of transversus abdominis indicates a deficit of motor control and is hypothesized to result in inefficient muscular stabilization of the spine.     

Coordinated expression of Hoxa-11 and Hoxa-13 during limb muscle patterning

The limb muscle precursor cells migrate from the somites and congregate into the dorsal and ventral muscle masses in the limb bud. Complex muscle patterns are formed by successive splitting of the muscle masses and subsequent growth and differentiation in a region-specific manner. Hox genes, known as key regulator genes of cartilage pattern formation in the limb bud, were found to be expressed in the limb muscle precursor cells. We found that HOXA-11 protein was expressed in the premyoblasts in the limb bud, but not in the somitic cells or migrating premyogenic cells in the trunk at stage 18. By stage 24, HOXA-11 expression began to decrease from the posterior halves of the muscle masses. HOXA-13 was expressed strongly in the myoblasts of the posterior part in the dorsal/ventral muscle masses and weakly in a few myoblasts of the anterior part of the dorsal muscle mass. Transplantation of the lateral plate of the presumptive wing bud to the flank induced migration of premyoblasts from somites to the graft. Under these conditions, HOXA-11 expression was induced in the migrating premyoblasts in the ectopic limb buds. Application of retinoic acid at the anterior margin of the limb bud causes duplication of the autopodal cartilage and transformation of the radius to the ulna, and at the same time induces duplication of the muscle pattern along the anteroposterior axis. Under these conditions, HOXA-13 was also induced in the anterior region of the ventral muscles in the zeugopod. These results suggest that Hoxa-11 and Hoxa-13 expression in the migrating premyoblasts is under the control of the limb mesenchyme and the polarizing signal(s). In addition, these results indicate that these Hox genes are involved in muscle patterning in the limb buds.     

Asymptomatic, unruptured carotid-ophthalmic artery aneurysms: angiographical differentiation of each type, operative results, and indications

Replication-defective (E1-E3-deleted) human adenovirus vectors are a promising means of therapeutic gene delivery to skeletal muscle cells. Since the tropism of adenovirus is nonselective, muscle-specific expression of systemically administered vectors can only be achieved by the use of a tissue-specific promoter/enhancer that is small enough to fit the insert capacity of the vector. We have generated two replication-defective adenovirus recombinants (AV) in which the reporter gene (either firefly luciferase or E. coli beta-galactosidase) was driven by a truncated (1.35 kb) muscle creatine kinase (MCK) promoter/enhancer or by the fast troponin I (TnI) promoter/enhancer. Highly efficient and muscle-specific transgene expression was demonstrated in immunodeficient mice after local injection of AV into muscles at an early age. In nonmuscle tissues (brain, liver, kidney, lung), the transgene expression was extremely low even though in these tissues in situ polymerase chain reaction showed as high an infectivity of the cells by the AV as in muscle. The relatively small size, the good efficiency and the muscle specificity of the MCK promoter would make it ideal to drive the 6.3 kb (truncated) dystrophin cDNA in first generation AV (with a limited (8 kb) insert capacity) designed for gene therapy of Duchenne muscular dystrophy.