Beyond the Identification of Transcribed Sequences:
Functional, Evolutionary and Expression Analysis
12th International Workshop
October 25-28, 2002
Washington, DC

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Full Domain Transcript of Alpha-Globin Genes is a Component of the Nuclear Matrix

A.V. Rynditch1, V.P. Smalko1, S.V. Razin2, E. Loudinkova2, K. Scherrer3
1Institute of Molecular Biology and Genetics, National Academy of Sciences of the Ukraine, Kiev, Ukraine; 2 Institute of Gene Biology, Russian Academy of Sciences, Moscow, Russia; 3 Institut J. Monod, 2, Place Jussieu, 75251 Paris, France

The investigation concerns the nature and possible significance of the Full Domain Transcript (FDT) of the alpha-globin genes in erythroleukaemic cells where globin gene expression is abortive. Using Northern blot hybridisation with strand-specific probes recognizing alpha-globin genes and intergenic sequences, we demonstrate that the entire locus of alpha-globin genes spanning ~ 30 kb between DNaseI hypersensitive sites is transcribed. The 3'-end of the transcribed area is mapped about 1500 bp downstream of alpha A, the last of the 3 alpha-type globin genes. Using primers downstream of the last gene and amplification of the cDNA with primer pairs placed way upstream, RT-PCR shows that about 30 kb of the sequenced domain in transcribed into one continuous FDT. This extends from the upstream control region over the replication origin and DNA loop anchorage site of the domain to the enhancer/silencer located downstream of the adult alpha A gene. RT-PCR and in situ hybridization with globin genic and extra-genic probes demonstrated the presence of such globin FDTs as a component of the nuclear matrix. On dividing AEV-transformed cells, FDTs were found to accumulate around the nucleoli, and to be excluded from those in matrix preparations. We propose that globin FDTs are the part of the dynamic nuclear architecture in the processing pathway and a component of the matrix, turning over slowly in AEV cells where globin gene expression is blocked post-transcriptionally.

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