LOVELY PROFESSIONAL

UNIVERSITY

Assignment Report
Submitted in partial fulfillment for the continuous
assessment of
Genetics (BTY 551)
For the degree of post-graduation of Biotechnology Hons.
2021.
Submitted By : Submitted to :
Tejaswi Harshwardhan Assisant Professor
Roll No: RB2108A88 Dr.Chirag Chopra
Registration No: 12114439
Q1.

Suppose you discovered a temperature‐sensitive mutant whose nucleus failed to accumulate

certain nuclear proteins at an elevated (restrictive) temperature but continued to accumulate
other nuclear proteins. What conclusions might you draw about nuclear localization and the
nature of this mutation?

ANS: Proteins to be transported into or out of the nucleus are bound by transport receptors that
recognize specific sequences in the cargo protein called nuclear targeting signals. The receptor cargo
complex is then translocated across the nuclear envelope and, following translocation, the complex
dissociates resulting in the delivery of the cargo to its appropriate compartment. Nuclear transport is
a highly regulated process with controls that dictate both if and when a cargo can enter and exit the
nucleus. Most mechanisms underlying the regulation of transport modulate the interaction of the
transport receptors with their cargo proteins. Disrupting this regulation can result in many negative
consequences to the cell and potentially to the entire no organism. All nuclear proteins and RNAs
are transported through the nuclear membrane via an active process controlled by the nuclear pore
complex . This transport requires activation by a NLS belonging either directly to the transported
molecule or to an associated helper molecule

It is given that at high temperature, one of the nuclear protein is not able to accumulate inside
nucleus. This is because of the mutation. Nuclear localisation signal sequence is present at the end
of 5’ end of dna. These are known as NPL mutants.

Q2.

The gene for a eukaryotic polypeptide 300 amino acid residues Iong is altered so that a signal
sequence recognized by SRP occurs at the polypeptide's amino terminus and a nuclear localization
signal (NLS) occurs internally, beginning at residue 150. Where is the protein likely to be found in
the cell?

ANS: We know that the protein is found in the Endoplasmic Reticulum. The protein from ER to be
targeted will depend on different signals. The Amino terminal is bind by the Srp, which signals entry
in protein synthesis and directs the nascent polypeptide and ribosome to receptor in the
endoplasmic reticulum.

Because the protein is translocated into the lumen of the ER as it is synthesised , the nuclear
localization signal(NLS) is never accessible to the protiens involved in nuclear targeting

In eukaryotes, SRP binds to the signal sequence of a newly synthesized peptide as it emerges from
the ribosome. This binding leads to the slowing of protein synthesis known as "elongation arrest", a
conserved function of SRP that facilitates the coupling of the protein translation and the protein
translocation processes. SRP then targets this entire complex (the ribosome-nascent chain complex)
to the protein-conducting channel, also known as the translocon, in the endoplasmic reticulum (ER)
membrane. This occurs via the interaction and docking of SRP with its cognate that is located in
close proximity to the translocon.
Q3.

Nuclear Speckles are the sites where splicing factors are concentrated. How is this localization
advantageous to the cells?

ANS: As we know,alternative pre-mRNA splicing greatly increases transcriptome diversity in higher

eukaryotes, nuclear bodies which are involved in splicing regulation are key gene expression
regulators. These bodies include nuclear speckles, which are also known as splicing speckles, B
snurposomes, splicing factor compartments, SC-35 domains and interchromatin granule clusters.
Several lines of evidence point to speckles acting as storage/assembly/modification compartments
that can supply splicing factors to active transcription sites

Many pre-mRNA splicing factors, including snRNPs and SR proteins , have been localized to nuclear
speckles by either immunofluorescence, fluorescent protein-tagging, and/or immunoelectron
microscopy. In fact, this speckled localization pattern is highly diagnostic for proteins involved in pre-
mRNA splicing. In addition, several kinases (Clk/STY, hPRP4, and PSKH) and phosphatases (PP1) that
phosphorylate/dephosphorylate components of the splicing machinery have also been localized to
nuclear speckles. This supports the idea that speckles may be involved in regulating the pool of
factors that are accessible to the transcription/pre-mRNA processing machinery.

Q4. Discuss an experiment in detail that you would plan to discover the sites for DNA replication in
a cell. The output of the experiment should be a visualization of these replication factories in the
cells. You have the liberty of choosing any system (insect cells/yeast/mammalian cells).

ANS: AIM OF THE EXPERIMENT: Analysis of replication factories in human cells by super-resolution
light microscopy

INTRODUCTION:

DNA replication in human cells is performed in discrete sub-nuclear locations known as replication
foci or factories. These factories form in the nucleus during S phase and are sites of DNA synthesis
and high local concentrations of enzymes required for chromatin replication. Why these structures
are required, and how they are organised internally has yet to be identified. It has been difficult to
analyse the structure of these factories as they are small in size and thus below the resolution limit
of the standard confocal microscope. We have used stimulated emission depletion (STED)
microscopy, which improves on the resolving power of the confocal microscope, to probe the
structure of these factories at sub-diffraction limit resolution.

PROCEDURE:

In order to visualise replication factories in human cells we selected antibodies against two key
components, PCNA and RPA. RPA is a heterotrimeric single stranded DNA (ssDNA)-binding protein
that associates with the template strands produced at replication forks by the action of the
replicative helicase. It is important for strand stability and for recruiting other replication
components to the advancing replication fork [30]. PCNA is a ringshaped sliding clamp protein which
is loaded onto template DNA at replication forks, where it acts as a platform for the recruitment of
multiple enzymes required for replication [31]. We first characterised the antibodies for
immunofluorescent detection of replication factories using standard confocal microscopy (figure 1).
MRC5 cells were pulse labelled with EdU to mark sites of DNA synthesis, then processed for
immunofluorescence. EdU was detected using the Click-it cell proliferation kit (Invitrogen). Proteins
were visualised indirectly using Atto 647N- or Alexa Fluor 488-linked secondary antibodies. As
previously demonstrated PCNA and RPA were localised in focal patterns in nuclei that were labelled
with EdU and thus actively replicating DNA . Throughout this study we selected cells that showed the
characteristic staining found in early S-phase, with many evenly sized nuclear replication factory
distributed evenly throughout the nucleoplasm . In these cells the PCNA and RPA patterns closely
follow that of EdU, and the colocalisation is striking but not complete . This is expected as PCNA is
loaded at primer-template junctions, and RPA binds template DNA, whereas the EdU is incorporated
as DNA is synthesised. Thus EdU labelled DNA can persist in locations after RPA or PCNA have
dissociated, and RPA or PCNA can bind to regions before DNA synthesis commences. We then used
the antibodies in combination to verify that the majority of these structures contained both RPA and
PCNA (figure 1C and 1D). Again the colocalisation analysis shows highly similar patterns, as expected
(supplemental figure S1 and table S1 [Additional file 1]). Some nuclear structures contain RPA but
not PCNA. We have not excluded these from our later analyses, it is possible that they are replication
factories that have recently initiated and do not yet contain active forks and PCNA, or that they are
other non-replication associated RPA regions.

further ‘’STED’’ method can also be used to visualize the replication factory on a better resolution.

Q5.

Sec61 is a protein involved in the formation of the translocon for ER-targeted proteins. What
would happen to the proteins targeted to the Golgi Apparatus in a mammalian cell in which the
Sec61 is mutated?

Ans: The Sec61 translocon on the ER membrane is a highly conserved multi-subunit protein complex
that consists of three subunits, Sec61α, Sec61β, and Sec61γ (of 476, 96, and 68 amino acid residues,
respectively) in eukaryotic cells. The pore of the channel is largely formed by the large Sec61α
subunit (or the homologous Sec Y subunit in prokaryotic cells). The Sec61 translocon is associated
with many other protein factors, for example, the Sec62/Sec63 proteins that are needed for the
posttranslational translocation. The Sec61 translocon complex forms a protein conducting channel
connecting the cytoplasmic and luminal spaces on either side of the ER membrane. The minimum
structure capable of translocating proteins through the ER membrane is a heterotrimer composed of
Sec61α, Sec61β and Sec61γ subunits, although these trimeric complexes have been observed to
dimerize.

The heterotrimeric Sec61 complex and the dimeric Sec62/Sec63 complex are located in the
membrane of the human endoplasmic reticulum (ER) and play a central role in translocation of
nascent and newly synthesized precursor polypeptides into the ER.

Protein secretion starts with protein translocation into the endoplasmic reticulum (ER) where
secretory proteins mature into a functional three-dimensional conformation before they are
packaged into ER-to-Golgi transport vesicles . Proteins that fail to fold in the ER are not allowed to
enter these vesicles, and are initially retained in the ER . Most are subsequently exported to the
cytosol and degraded by proteasomes, a process called ER-associated degradation (ERAD)
Hence due to mutation of sec 61 in golgi targeted proteins:

1. Because most of Sec61p is embedded in the membrane, mutagenesis of the entire SEC61 gene
predominantly leads to mutations in transmembrane domains

2. Translocon-associated protein” cant bind to precursor polypeptides in transit through the Sec61
channel and support their partial or complete translocation by acting as molecular ratchets