Stable (Heritable) DNA Transfer without Antibiotic Selection?

On Mendiburo et al., 2011 in Science

     Centromeres are the chromatin regions that are specialized to carry out the chromosome segregation during cell division. Because the spindle attachment sites are stable and inherited, initial analyses suggested that centromeres were sequence specific and the centromere formation is dictated by DNA sequence alone.  However, in very rare occasions centromeres can leave this stable spot and move to a new chromosomal site. These "neocentromeres" are as valid as natural centromeres and segregate the chromosomes with high fidelity.  This finding implies that the source of the specificity and the heritability of the centromeres might not be genetic but rather epigenetic.
A clue comes from the finding that specialized histone H3, called centromere protein A (CENP-A), is incorporated to the nucleosomes at centromeric loci. Mendiburo et al, hypothesized that this special histone could mark the centromeres on the chromosome. (Mendiburo et al., 2012) To test this they fused  CENP-A to bacterial LacI protein and engineered a LacO site  (which binds to the LacI) in their cell culture model. Surprisingly CENP-A was localized to the LacO and was incorporated to the local nucleosomes and recruited even the endogenous CENP-A to the new site.  This neocentromere was inherited and associated with spindle assembly normally. Although adding a second centromere causes mitotic failure and cell death, they also show that plasmids engineered like this can act as artificial chromosomes.
This brings up the idea that recombinant DNA transfected to eukaryotic cultured cells can be made heritable and stable without the need for heavy antibiotic selection and genome integration. Antibiotic selection is very strong and kills all the cells without the plasmid and the cells which looses the plasmid and only genome integrated copies remain. Some cells are fragile and do not survive this process. Another disadvantage is that genome integration is random and might cause aberrations if the integration site is vital. This is especially important in the case of gene therapy because genome integration could cause additional defects while mending the disease.

María José Mendiburo, 

Jan Padeken, Stefanie Fülöp,Aloys Schepers and Patrick HeunDrosophila CENH3 Is Sufficient for Centromere FormationScience 4 November 2011: 334 (6056), 686-690. [DOI:10.1126/science.1206880]

Long Non-Coding RNAs

On Ørom and Shiekhattar, 2011 in Curr Opin Genet Dev

Approximately 1.5 % of our genome is transcribed to protein-coding mRNA.  The existence of noncoding RNAs such as ribosomal and transfer RNAs as well as small nucleolar RNAs has been known for a long while now. More recently the RNA interference machinery and the associated microRNAs have been associated with important functions.  Now thanks to next-gen sequencing, it is becoming clear that remainder of the genome is not all junk but could code for long noncoding RNAs (long ncRNA or lncRNA).

Ørom and Shiekhattar give a review of the current research on these RNAs and they elaborate on the link with enhancers (Ørom and Shiekhattar, 2011). The authors note that the current understanding on long ncRNAs is very primitive. lncRNAs are very heterogenous which makes it hard to classify and decode the information they carry. Their distinctive property is that they lack protein-coding ORFs and they are longer than small regulatory RNAs .

Some of the complexities include changes in size and copy number (average length ~1kb).  Specific studies such as the ones on the HOX gene cluster and the beta-globin locus show that some lncRNAs can have regulatory functions on the expression of nearby protein coding genes. Some of the studied lncRNAs act in cis while others in trans which adds to the heterogeneity. In addition some of the lncRNAs have features of protein coding mRNAs such as polyAdenylation and splicing.

In both mouse and human cells, some lncRNAs are associated with enhancers. The elucidation of histone marks around the active enhancers supports this hypothesis. Although correlative analyses implicate these lncRNAs in the positive regulation of nearby genes, a mechanistic understanding is missing. What kind of code or information do lncRNAs have, to mediate their functions? This seems to be the foremost challange  in the field.  Their hetreogeneity and the possibility of multiple mechanisms granted, one hypothesis suggests that 3-dimensional structures of lncRNAs might interact with transcription factors and function to alter gene expression. However, published studies are not enough to reach a conclusion about this theory, yet.

Ulf Andersson Ørom, Ramin Shiekhattar, Long non-coding RNAs and enhancers, Current Opinion in Genetics & Development, Volume 21, Issue 2, April 2011, Pages 194-198, ISSN 0959-437X, 10.1016/j.gde.2011.01.020.