Parr T, Waites G T, Patel B, Millake D B, Critchley D R
Department of Biochemistry, University of Leicester, England.
Eur J Biochem. 1992 Dec 15;210(3):801-9. doi: 10.1111/j.1432-1033.1992.tb17483.x.
A chick non-muscle alpha-actinin cDNA probe encoding the EF-hand region of molecule was used to screen a lambda gt10 chick brain cDNA library from 14-day embryos. A partial 2.1-kb alpha-actinin cDNA was isolated (8W cDNA) which encoded a protein identical to chick skeletal-muscle alpha-actinin, except in the C-terminal part of the first EF hand. In the variant, the 22 residues found in the skeletal-muscle isoform were replaced by a stretch of 26 unique residues. Analysis of the structure of the skeletal-muscle alpha-actinin gene showed that the region of divergence was encoded by two exons which are alternatively spliced. Quantitative reverse transcriptase/polymerase chain reaction (RT/PCR) was used to investigate the levels of the alpha-actinin transcripts in various tissues. The skeletal-muscle alpha-actinin variant was expressed at low levels in brain, liver and spleen, but could not be detected in skeletal muscle. Surprisingly, skeletal-muscle alpha-actinin mRNA was also expressed in brain, liver and spleen. The RT/PCR products were authenticated by using diagnostic restriction enzyme sites and by sequencing. The splice variant derived from the skeletal-muscle alpha-actinin gene was also detected in a variety of cDNA libraries from both adult and embryonic tissues by PCR. Although a transcript encoding this alpha-actinin splice variant is expressed in non-muscle tissues, neither of the two EF-hands would be predicted to be functional, making it unlikely to be a typical non-muscle isoform which are calcium-sensitive with respect to binding actin. The two vertebrate non-muscle alpha-actinins sequenced to date also have a spacer of five amino acids between the two EF hands, whereas in the variant, the spacer is just four residues in length. Further analysis will be required before this alpha-actinin isoform, which we refer to as SKv, can be classified as muscle or non-muscle alpha-actinin. We propose a new nomenclature to describe the various alpha-actinin genes and their transcripts.
使用编码分子EF手区域的鸡非肌肉α-辅肌动蛋白cDNA探针，从14天胚胎的λgt10鸡脑cDNA文库中进行筛选。分离出一个部分2.1 kb的α-辅肌动蛋白cDNA（8W cDNA），其编码的蛋白质与鸡骨骼肌α-辅肌动蛋白相同，只是在第一个EF手的C末端部分有所不同。在该变体中，骨骼肌同工型中发现的22个残基被一段26个独特残基所取代。对骨骼肌α-辅肌动蛋白基因结构的分析表明，差异区域由两个外显子编码，这两个外显子可选择性剪接。采用定量逆转录酶/聚合酶链反应（RT/PCR）研究了α-辅肌动蛋白转录本在各种组织中的水平。骨骼肌α-辅肌动蛋白变体在脑、肝和脾中低水平表达，但在骨骼肌中未检测到。令人惊讶的是，骨骼肌α-辅肌动蛋白mRNA也在脑、肝和脾中表达。RT/PCR产物通过使用诊断性限制性酶切位点和测序进行鉴定。通过PCR在来自成年和胚胎组织的各种cDNA文库中也检测到了源自骨骼肌α-辅肌动蛋白基因的剪接变体。尽管编码这种α-辅肌动蛋白剪接变体的转录本在非肌肉组织中表达，但预计两个EF手中任何一个都不具有功能，这使得它不太可能是对结合肌动蛋白具有钙敏感性的典型非肌肉同工型。迄今为止测序的两种脊椎动物非肌肉α-辅肌动蛋白在两个EF手之间也有一个五个氨基酸的间隔区，而在该变体中，间隔区仅四个残基长。在将这种我们称为SKv的α-辅肌动蛋白同工型归类为肌肉或非肌肉α-辅肌动蛋白之前，还需要进一步分析。我们提出一种新的命名法来描述各种α-辅肌动蛋白基因及其转录本。
