Name : Devi Era Rachmawati

(175090101111005)

Class : Biology B

TASK

Housekeeping Genes

Housekeeping genes are some genes that required for the fundamental operations of
the cell and are therefore expressed under most conditition (Clark, 2005). As there is a vast
difference in how tissue-specific in contrast to housekeeping genes must be regulated, it is not
surprising that the promoters of these genes differ as well. Most housekeeping genesutilize a
promoter lacking the common TATA and CAAT boxes, and having instead a series of GC
boxes (consensus sequence GGGCGG). GC boxes provide binding sites for the transcription
factor Sp1 and, like the TATA box, direct the start of transcription. Since there are several GC
boxes in the promoters of many housekeeping genes, the transcription start site is ambiguous.
Indeed, many housekeeping gene transcripts have heterogeneous 5′ start sites. The coding
function of these genes is not impaired, however, because all of the alternative start sites are
within the 5′ untranslated region of the mRNA. As an example, the human c-Ha-ras oncogene
promoter has about 80% G+C content, 10 GC boxes, and at least four transcription start sites
(Goldman, 2001).

SDS (Sodium Dodecyl Sulfate)

SDS is a negatively charged (an ionic) detergent (some soap and shampoos). When
protein mixtures are boiled in SDS-containing buffers, they denature (unfold and assume a
more linear configuration). The SDS binds to the denatured protein quantitatively: the number
of SDS molecule bound is proportional to the number of amino acids in the protein
quantitavely: the number of SDS molecule bound is proportional to the number of amino acid
in th eprotein. The protein thus have an overall negative charge, irrespective of their native
charge(Corley, 2004).

(Clark, 2005)

Figure 1. Denaturation of Protein by SDS (A) The structure of SDS is amphiatic, that is, the
long hydrocarbon tail is hydrophobic and the sulfate is hydrophilic. (B) The first step to
seperate a mixture of proteins by size is to boil the sample. Boiling proten in a solution of
SDS destroy the tertiary structure of the protein. The hydropobic portion of SDS coats at
polypeptide backbone and prevents the protein from refolding. The hidrophilic group of SDS
keeps the protein soluble in water, and the negative charges repel each other, which also helps
to keep the protein from refolding. The result ia an unfolded protein with a net negative
charge that is porpotional to its molecular weight.

SDS-PAGE

SDS PAGE is one of the most widely used methods for analyzing proteins. Its
quantitative and separates proteins according to their size. SDS-PAGE usually used to
determine the molecular mass of protein s, to monitor protein purity and in conjuction with
western blot analysis to identify the presence of protein in complex mixtures (Corley, 2004).
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is a technique used
to move charged molecules through a gel matrix by means of an electric current. This
procedure is used to determine protein subunit composition, verify homogeneity of the protein
sample, and purify proteins for use in other applications. The migration rate of the proteins
during SDS-PAGE is determined by the pore size of the gel matrix and charge, size, and
shape of the protein (Manns, 2011).

PAGE (Polycrylamide gel electrophoresis) is the gel that used in SDS-PAGE. The use
of PAGE gelas have had a major impact on our abillity to resolve proteins in complex
mixture, estimate their size, and determine some of their properties, it is the standard methods
in biomedicine. PAGE gels are remarkably versatile for performing high resolution separation
of proteins and nucleic acid (such as for resolving DNA ladders in DNA sequencing reaction,
or for RNase protection assays). PAGE gels can be formed in tubes or as a vertical “slab”
gels, which are formed and run between two glass plates. PAGE gels are referred to by the
percentage of acrylamide in the gels (4% PAGE gel 10 % PAGE gel, etc.). The percent
acrylamide is various deppending on the nature size of the macromolecule being separated
and the method of separation used. In genereal, lower percentage gels are used for resolving
necleid acid or for isoelectric focusing. In which protein are separated based on charge
characteristics. Higher percentage gels (10% or greater) are normally used for separating
protein in SDS-PAGE (Corley, 2004).

Protein separation by SDS-PAGE can be used to estimate the relative molecular mass
of proteins, the relative abundance of proteins in a sample, and the distribution of proteins
among fractions, as well as the purity of protein samples. SDS is an anionic (negatively
charged) detergent that can dissolve hydrophobic molecules and denature secondary and
nondisulfide-linked tertiary structures. Proteins solubilized in SDS have only primary
structure, and a large negative charge in proportion to the mass of the protein which
determines that they will migrate toward the anode (positive pole) of an electric field.
Polyacrylamide, a polymer of acrylamide monomers, forms a gel matrix which serves as a
sieve, slowing the rate of migration of larger molecules to a greater extent than that of smaller
molecules. Proteins migrate through the pores in the matrix in response to an electrical field.
The size of the pores is determined by the concentration of the acrylamide. The higher the
acrylamide concentration, the smaller the pore size in the gel matrix. The migration rate of the
protein is determined by the gel pore size as well as charge, size, and shape of the protein.
After a set amount of time, depending on the voltage across the gel, the proteins will
differentially migrate; smaller proteins will travel farther down the gel while larger proteins
remain closer to the point of origin (Maizel, 2000).

Figure 2. Sample SDS-PAGE of various protein

SAM

S-S-Adenosyl Methionine (SAMe), is a metabolit present in a living cells, tahat play a

central role in cellular biochemistry as a precusor to methylation, aminoprophilation, and
transfusion pathways. To sustain the normal function of these pathways, a sufficient source of
SAMe is essential. The amount of SAMe that is required by the body daily depends on the
availability of methionine produced by de novo synthesis (involving methyltretrahydrofolate
and vitamin B-12) and also methionine obtained from the diet, mainly from the breakdown of
proteins (Bottigliei, 2002).

(Bottigliei, 2002)

Figure 3. Metabolism of S-adenosylmethionine (modified with permission from

reference 12). SAMe, S-adenosyl-l-methionine; SAH, S-adenosylhomocysteine; Hcy,
 homocysteine; MTHF, methyltetrahydrofolate; THF, tetrahydrofolate; GSH,
glutathione; deca-SAMe, decarboxylated S-adenosyl-l-methionine; MTA,
methylthioadenosine; ATP, adenosine triphosphate; PPi, pyrophosphate.

cDNA (Complementary DNA)

Complementary DNA (cDNA) is the DNA produced on an RNAtemplate by the

action of reverse transcriptase (RNA-dependent DNA-polymerase). The sequence of
the cDNA becomes complementary to the RNA sequence. Unlike RNA, DNA molecules can
be cloned easily (these are called „cDNA clones‟) by making the cDNA double-stranded and
ligated to a vector DNA. Sequence analysis of DNA is much easier than that of RNA, thus,
cDNA is the essential form in the analysis of RNA, particularly of eukaryotic mRNA
(Kohara, 2001).

(Kohara, 2001).
Figure 3. Synthesis of cDNA

cDNA synthesis includes a two-step (first- and second-strand synthesis) conversion of

the cleaned up RNA to more stable copy DNA (cDNA). For this purpose the specialized
enzyme reverse transcriptase is utilized, which converts single-stranded RNA (ssRNA) into
DNA. For microarray sample preparation the first-strand synthesis begins by reverse
transcription with an oligo(dT) primer, which anneals efficiently to the poly(A) tails of
the mRNA. It is exactly this step that also allows the application of total RNA, since only
those RNA molecules containing the poly(A) tail will be further processed, ie, the mRNA.
The starting material for the second-strand synthesis is mRNA–cDNA hybrid molecules.
Before the DNA polymerase can synthesize the second strand of the cDNA, RNase H is
added to the mRNA–cDNA hybrid transcripts. RNase H digests only the ss mRNA at
unspecific sites, thus creating short mRNA fragments that remain bound to the ss cDNA and
are utilized by the DNA polymerase as primers for the second-strand synthesis. The resulting
material, which will be further processed, is double-stranded cDNA (ds cDNA) (Kohara,
2001).
 REFFERENCE

Bottigliei, Teodoro. 2002. S-Adenosyl-L-methionine (SAMe): from the bench to the

bedside molecular basis of a pleiotrophic molecule. The Merican Journal of Clinical
Nutrition. Vol. (76) No. (5)
Corley, Ronald B. 2004. A Guide to Methods in the Biocmedical Science. Springer. Boston.

Kohara, Y. 2001. cDNA. Encyclopedia of Genetics. Page 286

Manns, Joanne M. 2011. SDS-Polycarmide Gel Electhrophoresis (SDS PAGE) of Protein.

Current Protocols in Microbiology.