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874 Nucleic Acid Synthesis

2-nm-wide naked DNA fiber to metaphase chromosomes mRNAs of several kinetoplastid species (Crithidia, Try-
of microscopic width. The DNA template has to be ac- panosoma, etc.) were found to be edited by the insertion
cessible to transcription machinery containing RNA poly- and deletion of U’s at many sites in mRNAs. The editing
merase; transcriptionally inactive, highly compacted chro- processusesatemplateconsistingofaguideRNA(gRNA)
matin maintains its structure by multiple protein–protein whose genes function as independent transcription units.
and protein–DNA interactions, which are yet to be elu- The gRNAs are generally 55–70 nucleotides in length and
cidated. However, it is now clear that at the nucleosome complementary to the mRNA for a significant distance
level, it is the strength of interaction between histones including and surrounding the edited region. The gRNA
and DNA which regulates accessibility of the DNA to the dictates the specificity of uridine insertions by its pairing
transcription machinery, a process controlled by acetyla- with the pre-edited RNA, but also provides the U residues
tion and phosphorylation of core histones. Multiple his- that are inserted into the target RNA by transesterification
tone acetylases and deacetylases, which are themselves reactions; the reaction proceeds along the pre-edited RNA

regulated, modulate chromatin structure. As stated pre- in the 3 -5 direction. The RNA editing process reveals the
viously, large protein complexes named SWI and SNF existence of a previously unrecognized level for the con-
modulate chromatin structure in an energy-dependent pro- trol of gene expression. Recognition of this process has
cess which may be responsible for the differentiation/ resulted in an expansion of the central dogma. Multiple
development-dependent turning on or off of specific sets RNA editing processes play a significant role in normal
of genes. physiological processes, as well as being responsible for
some disease.

2. CpG Methylation-Dependent Negative
Regulation of Genes
VII. CHEMICAL SYNTHESIS OF NUCLEIC
In addition to histone modification, DNA itself was found ACIDS (OLIGONUCLEOTIDES)
to be modified, most commonly by methylation at the
C-5 position of cytosine, but only when it is present as Development of strategies for chemical synthesis of nu-
a CpG dinucleotide. Such methylation, catalyzed by spe- cleic acids represented a major breakthrough in molecular
cific methyltransferases, invariably inhibits gene expres- biology, because most of the current approaches involving
sion, which was unequivocally established in the genomes PCR, manipulation of recombinant DNA, studies of gene
during embryonic development. Sets of genes are selec- regulation, etc. require synthetic DNA and RNA oligonu-
tively methylated or demethylated in the CpG sequences, cleotides with defined sequences. The difficulty of syn-
most commonly in the genes’ promoter regions, leading to thesizing RNA and DNA polynucleotide chains from
their activation or repression. Proteins that bind to methy- mononucleotide units lies in the reactivity of the side
lated CpG sequences have been implicated in the control chains of the bases and the susceptibility of the sugar
of histone deacetylation, thereby leading to closing of the glycosyl bond to cleavage under the harsh conditions
promoter. needed for condensation reactions to generate phospho-
diester bonds. An additional problem in RNA synthesis is
the presence of the C 2-OH group in ribose.

F. Fidelity of Transcription (RNA Editing)
H. Khorana’s group was the first to solve the problem
The informational content of gene transcripts can be al- by blocking all reactive side chains of the bases with re-
tered during or after transcription by a process collectively versible blocking groups; a phosphodiester bond between

called RNA editing. The information changes are carried C 3-OH of one nucleotide and the C 5-phosphate of ano-
out at the level of mRNA. RNA editing appears to be ther was generated by condensation in the presence of di-
a widespread phenomenon for both normal and aberrant cyclohexyl carbodiimide (DCC) under mild conditions.
RNA processing in organelles and nuclei. It was first dis- Repeating the process in a cyclic fashion generated
covered in the mitochondria of kinetoplasts in protozoa. oligonucleotides of a defined sequence. While the DCC
Two types of RNA editing have been observed: (1) al- condensation was efficient, the whole process was ex-
teration of coding sequence by nucleotide insertion and/ tremely laborious, because the products of each reaction
or deletion and (2) base substitution. In mammalian cells, had to be purified free of the side products and the blocking
editing of an individual base in mRNA can cause a change groups had to be removed after each cycle. Furthermore,
in the sequence of the protein. Such changes can occur by the efficiency of the synthetic reaction fell off rapidly with
enzymatic deamination in which C is converted to U or increasing size of the oligonucleotide.
A is converted to hypoxanthine. Change of U to C has A major advance occurred in the 1970s when two dis-
also been observed in many plants. The (mitochondrial) tinct types of chemistries were invented for synthesis of