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Online Counseling Resource
YCMOU ELearning Drive…
School of Architecture, Science and Technology
Yashwantrao Chavan Maharashtra
Open University, Nashik – 422222, India
OC-SBT053-CP1-03
Introduction
Programmes and Courses
 SEP – SBT053 – Unit 01
School of Science and Technology, Online Counseling Resource…
Credits
 Academic Inputs by
 Mrs. Rasika Bhore
 M.sc (Microbiology)
 [email protected]
© 2007, YCMOU. All Rights Reserved.
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School of Science and Technology, Online Counseling Resource…
How to Use This Resource

Counselor at each study center should use this presentation to deliver
lecture of 40-60 minutes during Face-To-Face counseling.

Discussion about students difficulties or tutorial with assignments should
follow the lecture for about 40-60 minutes.

Handouts (with 6 slides on each A4 size page) of this presentation should
be provided to each student.

Each student should discuss on the discussion forum all the terms which
could not be understood. This will improve his writing skills and enhance
knowledge level about topics, which shall be immensely useful for end
exam.

Appear several times, for all the Self-Tests, available for this course.

Student can use handouts for last minutes preparation just before end
exam.
© 2007, YCMOU. All Rights Reserved.
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School of Science and Technology, Online Counseling Resource…
Learning Objectives
 After studying this module, you should be able to:
 Discuss the role of Alkaline Phosphatase &
Reverse Transcriptase.
 Describe how they are use in Recombinant DNA
Technology & in research work.
© 2007, YCMOU. All Rights Reserved.
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School of Science and Technology, Online Counseling Resource…
Introduction
 Alkaline Phosphatase(ALP) is an important
enzyme exist in all human tissues as well as in
bacterial tissues.
 Elevation or decrease in ALP levels produce the
diseased condition.
 It also plays an important role in research.
 Reverse Transcriptase is a trademark of the
Retrovirus family.
 RT due to its unique property is very powerful
enzyme for viruses as well as in genetic
research.
School of Science and Technology, Online Counseling Resource…
Alkaline Phosphatase
 Alkaline phosphatase (ALP) is a
hydrolase enzyme responsible for
dephosphorylation means from many
types of molecules, including nucleotides,
proteins, and alkaloids.
 As the name suggests, alkaline
phosphatases are most effective in an
alkaline environment.
School of Science and Technology, Online Counseling Resource…
Bacterial Alkaline Phosphatase
 Alkaline phosphatase located in bacterial periplasmic
space is resistant to inactivation, denaturation, and
degradation, and also has a higher rate of activity.
 Alkaline phosphatase is produced by the bacteria only
during phosphate starvation and not when phosphate is
plentiful suggests, it generate free phosphate groups for
uptake and use.
 It also suggets that presence of phosphate groups
usually prevents organic molecules from passing
through the bacterial membrane, therefore
dephosphorylating them may be important for bacterial
uptake of organic compounds in the wild.
 In the laboratory, however, mutant Escherichia coli
lacking alkaline phosphatase survive quite well than the
mutants unable to shut off alkaline phosphatase
production.
School of Science and Technology, Online Counseling Resource…
Human Alkaline Phosphatase
 In humans, alkaline phosphatase is present in
all tissues throughout the entire body, but is
particularly concentrated in liver, bile duct,
kidney, bone, and the placenta.
 The optimal pH for the enzyme activity is
pH=10.
School of Science and Technology, Online Counseling Resource…
Diagnostic Use of Human ALP
 Blood plasma (serum) levels of ALP are typically 2070 units per liter in normal adults.
 Elevated levels (hyperphosphatasemia) cause :
 (Liver ALP): Cholestasis, cholecystitis, cholangitis,
cirrhosis, hepatitis, fatty liver, liver tumor, liver
metastases, drug intoxication etc.
 (Bone ALP): Paget's disease, osteosarcoma, bone
metastases of prostatic cancer, Other bone
metastases, Fractured bone, Multiple myeloma etc.
 (Skeletal diseases): Osteomalacia, rickets, vitamin
D deficiency, Malignant tumors, Renal disease etc.
School of Science and Technology, Online Counseling Resource…
Hypophosphatasemia
 Lowered levels of ALP are less common than
elevated levels.
 Hypophosphatasemia causes:
 Postmenopausal women – ostreoporosis
 Man with recent heart surgery –
malnutrition, severe anemia
 Children – achondroplasia, cretinism
School of Science and Technology, Online Counseling Resource…
Industrial Use of ALP
 It is used in the dairy industry as a marker of
pasteurisation.
 ALP is denatured by elevated temperatures
found during pasteurisation,which can be tested
via colour change of a para-nitro-phenol
phosphate substrate in a buffered solution.
 Raw milk would typically produce a yellow
colouration within a couple of minutes, whereas
properly pasteurised milk should show no
change.
 There are of course exceptions to this in the
case of heat stable alkaline phophatases
produced by some bacteria.
School of Science and Technology, Online Counseling Resource…
Use of ALP In Research
 Alkaline phosphatase has become a useful tool in
molecular biology laboratories.
1.
Problem arises when the cohesive ends of broken plasmids,
instead of joining with foreign DNA, join the cohesive end of
same DNA & get recircularized.
 To overcome this problem, the restricted plasmid is
treated with alkaline phosphatase,that digests the
terminal 5’ phosphoryl group.
2. Removal of the phosphate groups allows radiolabeling in
order to measure the presence of the labeled DNA through
further steps in the process or experiment.
 For these purposes, the alkaline phosphatase from shrimp
is the most useful, as it is the easiest to inactivate once it
has done its job.
3. Another important use of alkaline phosphatase is as a label
for enzyme immunoassays.
School of Science and Technology, Online Counseling Resource…
List of ALPs Used In Research
 The most common alkaline phosphatases used in
research are:
1. Bacterial alkaline phosphatase (BAP), from
Escherichia coli C4 cells.
2. Shrimp alkaline phosphatase (SAP), from a
species of arctic shrimp (Pandalus borealis).
3. Calf intestine alkaline phosphatase (CIAP), from
calf intestine.
4. Placental alkaline phosphatase (PLAP) and its C
terminally truncated version that lacks the last 24
amino acids (constituting the transmembrane
domain) - the secreted alkaline phosphatase
(SEAP).
School of Science and Technology, Online Counseling Resource…
Reverse Transcriptase
 Reverse transcriptase (RT) was discovered by Howard
Temin and independently by David Baltimore in 1970.
The two shared the 1975 ‘Nobel Prize in Physiology
or Medicine’ with Renato Dulbecco for their discovery.
 Reverse transcriptase is a common name for an enzyme
that functions as a RNA-dependent DNA polymerase.
 They are encoded by retroviruses, where they copy the
viral RNA genome into DNA prior to its integration into
host cells.
 These enzymes transcribes single-stranded DNA from
double-stranded DNA.
 This complementary strand of DNA integrates into host
cell DNA, leads to cause disease.
School of Science and Technology, Online Counseling Resource…
Commonly Used Reverse Transcriptase
1. HIV-1 reverse transcriptase from the
human immunodeficiency virus type 1
(PDB 1HMV).
2. M-MLV reverse transcriptase from the
Moloney murine leukemia virus.
3. AMV reverse transcriptase from the
avian myeloblastosis virus.
4. Telomerase reverse transcriptase that
maintains the telomeres of eukaryotic
chromosomes.
School of Science and Technology, Online Counseling Resource…
Process of Reverse Transcription
 Reverse
transcription
is the
process of
making a
double
stranded
DNA
molecule
from a single
stranded
RNA
template.
School of Science and Technology, Online Counseling Resource…
Structure of HIV-1 RT
 The enzyme is a
heterodimer
consisting of a full
66kDa RT monomer
complete with RNaseH
domain, and a 51kDa
RT monomer from
which the RNaseH
domain has been
cleaved.
 The 66kDa monomer
has identifiable
'finger', 'palm', and
'thumb' components.
School of Science and Technology, Online Counseling Resource…
Structure of HIV-1 RT
 The interface of the 66kDa and
51kDa monomers provides a track
from the polymerase active site to
the RNaseH active site, and allow for
the concomitant destruction of the
RNA primer template strand directly
after it has been copied.
 In a complex of RT with a double
stranded DNA molecule, the DNA
close to the polymerase active site is
in the A-form, while the rest is in the
normal B-form.
 The 51kDa monomer may also
provide a binding site for the lysinetRNA that primes initial DNA
synthesis.
School of Science and Technology, Online Counseling Resource…
Applications of RT
 Reverse transcriptase is commonly used in research to
apply the PCR technique to RNA called ‘reverse
transcription polymerase chain reaction’ (RT-PCR).
 The classical PCR technique can only be applied to DNA
strands, but with the help of reverse transcriptase, RNA
can be transcribed into DNA, thus making PCR analysis
of RNA molecules possible.
 Reverse transcriptase is also used to create cDNA
libraries from mRNA.
 The commercial availability of reverse transcriptase
greatly improved knowledge in the area of molecular
biology as, along with other enzymes, it allowed
scientists to clone, sequence and characterize DNA.
School of Science and Technology, Online Counseling Resource…
What We Learn………….
 ALP is an enzyme which removes the phosphate
group from DNA or RNA, hence used in genetics
experiment.
 ALP present in outer membrane of bacteria
supposed to helps to satisfy the phosphate
need of bacteria.
 Reverse Transcriptase catalyzes the synthesis
of DNA from RNA & insert it into host DNA.
 It is used for constructing cDNA libraries & can
make possible the PCR analysis of RNAs.
School of Science and Technology, Online Counseling Resource…
Critical Thinking Questions
• Why the antibodies fails to fight against
the HIV?
© 2007, YCMOU. All Rights Reserved.
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School of Science and Technology, Online Counseling Resource…
Tips For Critical Thinking Questions
 RT enzyme comprises complementary
DNA from RNA, the enzyme not always
read the RNA sequence correctly leads to
mutation in progeny.
© 2007, YCMOU. All Rights Reserved.
School of Science and Technology, Online Counseling Resource…
Study Tips
 Book

Title: Human Alkaline
Phosphatase

Author: A.R.Liss
 Book

Title: Biochemistry

Author: Dr. Mrs. Pankaja
Naik
• www.en.wikipedia.org
Reverse Transcriptase/ Alkaline
Phosphatase
• www.cellsalive.com
HIV infection: Reverse
Transcription.
• www.drkaslow.com
Alkaline Phosphatase
© 2007, YCMOU. All Rights Reserved.
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End of the Presentation
Thank You