1. Identification of Full-Length mysRS Element
Dishita Shah
Faculty Advisor: Dr. David Kass
Eastern Michigan University School of Biological Science
Abstract
Mys represents a family of retrotransposon, they change their position in the genome via an RNA intermediate. Upon investigating the evolution and origin of mys elements in rodents of the family Cricetidae, our lab generated an intra-mys PCR
genomic DNA library of the Mexican volcano mouse, Neotomodon alstoni. Based on alignment of DNA sequences, a similar but distinct family of elements was identified that we refer to mysRS (mys Related Sequence). Since this element has not
been characterized, we are utilizing a technique referred to as high-efficiency thermal asymmetric interlaced (hiTAIL) PCR in order to determine a full-length element utilizing DNA from P. maniculatus and N. alstoni. Identification of a full-length mysRS
element will provide unique insights regarding the origins, evolution, and genetic impacts of retrotransposons limited to a small taxonomic group of mammals.
Introduction
Mys element represent the family of retrovirus-like elements (RLEs) identified in
the white-footed mouse, Peromyscus leucopus. RLEs are one of the subclasses of
transposable element (TE), or “jumping genes” that can change its position in the
genome thus can create or revert mutation. Retrotransposons replicate and move from
one location to another via RNA intermediates therefore when active can alter the size
of genome. Their random integration can affect the stability of that site in chromosome,
DNA methylation potential of that site, and thus have potential role in evolution of
species.
The purpose of this study is to identify and characterize full-length of mysRS element
which will further improve the understanding of its origin, evolution and genetic
impacts on rodents.
Methods
• hiTAIL-PCR method used for amplification of mysRS element.
• PCR amplified products were analysed using 1% agarose gel.
• PCR products containing mysRS element were ligated and cloned using pGEM-T Easy
cloning vector and JM109 high efficiency competent cells.
• DNA was purified from the clones and checked for correct insert.
• DNA samples were then sequenced.
Current work
mysRS sequences are being analyzed and compared to mysRS
sequence identified in N. alstoni. Database search is being done to
further investigate mysRS sequences.
Acknowledgement
I would like to thank Dr. Kass for all his guidance and support for this project.
mysRS element
Figure-6. Primary hiTAIL-PCR amplified mysRS
element products using mysRS specific primers
mysRS1c 430F and ACI from P. maniculatus and
N. alstoni genome with different dilutions.
Lanes: 1. Middle range ladder DNA, 2. N. alsotni
(Na) 1:10, 3. P. maniculatus (Pm) 1:10, 4.
Control 1:10, 5. Na 1:100, 6. Pm 1:100, 7.
Control 1:100, 8. Na 1:1000, 9. Pm 1:1000, and
10. Control 1:1000.
Figure -7. Restriction digestion gel of the clones
that showed mysRS element insert and were sent
for sequencing. Lanes: 1. Middle range ladder,
2-6. Clones from N. alstoni, and 7-10. Clones
from P. maniculatus.
Figure-1. Transposition of retrotransposons
1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10
P. maniculatus (Deer mouse)
N. Alstoni (Mexican volcanic mouse)
Figure 2. Displays the results when sequencing the mys element.
The mys Neo 1 and 5 represent the mysRS element. The
differences between the two elements are highlighted in the
white boxes. From these differences the primers for the mysRS
element were designed.
Figure-4. Full length mys element
Results
Primary hiTAIL-PCR amplification with primers ACI and mysRS1c430F
mysRS element in both P. maniculatus and N. alstoni were amplified as
seen in figure-2. Clones generated using this PCR products with correct
size of insert were obtained as seen in figure-3 and then were sequenced.
Results
Marsh Rice Rat
Hispid Cotton Rat
Tumbala Climbing Rat
White-throated Wood
Rat
Fulvous harvest Mouse
Northern Grasshopper
Mouse
P. californicus
P. eremicus
P. gossypinus
P. leucopus
P. maniculatus
N. alstoni
P. aztecus
Reithrodontomini:
Location where
mysRS
element originated
Figure-3. Phylogenetic relationship between the rodent species that are being
compared with this study, and the possible location of the origin of the mysRS
element during evolution of the rodent genome, based on preliminary data.
Neotominae
Deer Mouse & Relatives
Genome ORF1 Genome
mysRS Element
SP1 SP2 SP3
AC1 LAD
Figure-5. hiTAIL-PCR amplification of mysRS element using ORF1 specific primers. LAD: universal
primer used during pre-amplification cycle. AC1: primer specific to LAD, used in primary hiTAIL cycle.
SP1, sp2 and SP3: primers specific to ORF region used in pre-amplification, primary cycle and secondary
cycle respectively.