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GENETIC MATERIAL
Several lines of indirect evidence have long suggested that DNA contains the gen
etic information of the living organism. Results obtained from several different
experimental procedures showed that most of the DNA is located in the chromosom
es, whereas the RNA and proteins are also abundant in the cytoplasm.
Moreover, a precise correlation exists between the amount of DNA per cell and th
e number of sets of chromosomes per cell. That is, most somatic cells of diploid
organisms for example, contain exactly twice the amount of DNA as the haploid g
erm cells or gametes of the same species. The molecular composition of the DNA i
n all the different cells of an organism is the same, whereas the composition of
RNA and protein varies both qualitatively and quantitatively from one cell type
to another.
Direct evidence showing that the genetic material is DNA rather than protein or
RNA was published by Avery, MacLeed and McCarty in 1944. They demonstrated that
the component of the cell responsible for the phenomenon of transformation in th
e bacterium Diplococcus pneumonia was DNA.
The genetic information of all living organisms, except the RNA viruses, are sto
red in DNA. Nucleic acids, originally called 'nuclein' because they were isolate
d from cell nuclei by F. Miescher, in 1869, are macromolecules composed of repea
ting subunits called nucleotides.
Each nucleotide is composed of
(1) a phosphate group,
(2)a five-carbon sugar, and
(3) a cyclic nitrogen are commonly found in DNA: adenine, guanine, thymine, and
cytosine.
RNA also usually contains adenine, guanine, and cytosine but has a different bas
e, uracil, in place of thymine. Adenine and guanine are double-ring bases called
purines; cytocine, thymine, and uracil are single ring bases called pyrimidines
. Hence both DNA and RNA contain four different subunits or nucleotides two puri
ne nucleotides and two pyrimidine nucleotides. RNA usually exists as a single st
randed polymer composed of a long sequence of nucleotides. DNA, however, has one
very important additional level of organization; it is usually a double strande
d molecule.
The correct structure of DNA was first deduced by Watson and Crick in 1953, as
a double helix in which the two polynucleotide chains are coiled about one anoth
er in a spiral. Each polynucleotide chain consists of a sequence of nucleotides
linked together by phosphodiester bonds, joining adjacent deoxyribose moieties.
The two polynucleotide strands are held together in their helical configuration
by hydrogen bonding between bases in opposing strands, the resultant base pairs
being stacked between the two chains perpendicular to the axis of the molecule l
ike the steps of a spiral staircase. The base pairing is specific adenine is alw
ays paired with thymine and guanine is always paired with cytosine. Thus, all ba
se pairs consist of one purine and one pyrimidine.
Once the sequence of bases on one strand of a DNA in a double helix is known, th
e sequence of bases in the other strand is also known because of specific base p
airing. The two strands of a DNA in a double helix are thus said to be complemen
tary.
However, the structure of DNA molecules changes as a function of their environm
ent. The exact conformation of a given DNA molecule or segment of a DNA molecule
will depend on the nature of the molecule with which it is interacting.
Certain DNA sequences have been shown to exist in a unique, left handed double h
elical form called Z-DNA (Z for the zigzag path of the sugar phosphate backbones
of the structure). In a high concentration of salts or in a dehydrated state DN
A exists in the A-form which has 11 nucleotide pairs per turn.
It is very unlikely that DNA molecules ever exist in the A-form in VIVO. The hel
ices of the A- and B-forms of DNA are wound clockwise. They can undergo conforma
tional shifts from the B-form to the Z-form and vice versa. Certain regulatory p
roteins are bound to the Z-form or B-form of a DNA sequence and cause it to shif
t to the B-form or Z-form.
DNA CONTENT
Plant cells contain large amounts of DNA, the amount being very variable between
species from the smallest (Arabidopsis thaliana, 0.5 picogram (pg) per haploid
genome) to some members of Loranthaceae (with over 100 pg per haploid genome). E
ven the smallest plant genome is about five times larger than that found in Dros
ophila melanogaster and contains much more DNA than is required to specify all t
he proteins synthesized during the course, of development.
This sequence, or one very similar to it, occurs at about nucleotide -30 (30 nuc
leotides upstream from the cap site) in almost all plant and animal genes. This
sequence is required for correct expression of eukaryotic genes in vitro and acc
urate, efficient initiation in vivo.
Since plant genes are also transcribed by RNA polymerase it is not surprising th
at all the plant genes have a sequence analogous to the TATA box. The distance b
etween the TAT A box (measured from the second T) and the cap site is 29-33 nucl
eotides, as in other eukaryotes.
AGGA BOX
Another sequence that may be involved in regulation of transcription of some euk
aryotic genes is the consensus sequence "CAAT" box or GG(CT)CAATCT. This sequenc
e is often found -40 and -180 nuleotide upstream of the cap site. The sequence o
f this element is much more variable but often includes the sequence CAAT, terme
d the "CAAT Box". In plants it is referred to as the AGGA box. Two zein genomic
clones, Z4 and Z7, have sequences with limited homology to the CAAT box. The leg
hemoglobin genes have three sequences upstream of the coding region, all with so
me homology to the animal sequence. All of plant sequences have an interesting s
ymmetry of adenines surrounding the trinucleotide (G/T)NG.
OTHER REGULATORY ELEMENTS
Other regulatory elements also occur in this region. In general, these elements
seem to modify the promoter specificity, making transcription of the gene respo
nsive to particular environmental signals. Promoter specificity can often be dem
onstrated by attaching promoter containing sequences of one gene to the coding s
equences of another and introducing the resultant hybrid gene into cells in whic
h it can be expressed.
Although a few cases are known wherein regulatory elements occur in the gene its
elf (a prime example is 5S rRNA genes transcribed by RNA polymerase III), transc
riptional responses usually seem to be determined by the sequences5' to the star
t of transcription, often called 5'-flanking sequences.
A good example is provided by the heat shock genes which are activated when cell
s are subjected to thermal stress. In both animals and plants it has been Possib
le to show that the 5' flanking sequences from a heat shock gene can confer resp
onsiveness to a gene not normally responsive to heat shock. This element seems t
o be required for responsiveness to heat shock and is located in a region which
can be shown by direct binding experiments to interact with a protein transcript
ion factor. For example, in Drosophila the flanking sequences of different heat
shock genes contain a common element that is 14 nucleotides long and located 11-
28 bases upstream from the TATA box.
Another most interesting class of control elements is the enhancer sequences. Th
ese elements were originally discovered in viral genes, in which they were shown
to be required for high levels of transcription. An "enhancer core" sequence su
ch as GGTGTGGAAAG, or more generally GTGGT/AT/AT/AG ('II A' means that either T
or A is found in this position), can be identified by sequence comparisons, alth
ough it is clear that sequences other than the core are also important.
Enhancer sequences are often associated with regions of alternating purines and
pyrimidines which are capable of forming Z-DNA. Enhancers like sequences in plan
ts stimulate the expression of genes such as RuBP carboxylase in cells grown in
light but not those grown in the dark.
REPEATED SEQUENCES OF REGULATING GENE EXPRESSION
In a strict sense, some of the control sequences just described is really repeat
ed sequences. Short elements, such as the TATA or CAAT boxes, appear in many pro
moters and hence are repeated, but because they are so short are not recognized
in hybridization experiments.