Biomedical Engineering Reference
In-Depth Information
Target DNA
Eco
R
I
cloning site
CEN4
ARS1
URA 3
TRP1
pYAC vector
AMP
ori
TEL
TEL
Bam
H
I
Bam
HI
Bam
H
I
and
Eco
R
I
-digest
Partial
Eco
R
I
-digest
TRP
ARS
CEN
Fig. 9.3
Construction of a yeast
artificial chromosome containing large
pieces of cloned DNA. Key regions of the
pYAC vector are as follows: TEL, yeast
telomeres; ARS 1, autonomously
replicating sequence; CEN 4,
centromere from chromosome 4;
URA3
and
TRP1
, yeast marker genes; Amp,
ampicillin-resistance determinant of
pBR322;
ori
, origin of replication of
pBR322.
URA
L
igat
e
TEL
TRP
ARS
CEN
URA
TEL
ORF 1. Adams
et al
. (1987) have shown that fusion
proteins can be produced in cells by inserting part of
a gene from human immunodeficiency virus (HIV)
into ORF 1. Such fusion proteins formed hybrid
HIV:Ty-VLPs.
The Ty element can also be subjugated as a vector
for transposing genes to new sites in the genome.
The gene to be transposed is placed between the 3
LTR
LTR
Primary transcript
ORF 1
ORF 2
Fig. 9.4
Structure of a typical Ty element. ORF 1 and ORF 2
represent the two open reading frames. The delta sequences
are indicated by LTR (long terminal repeats).
′
end of ORF 2 and the 3
delta sequence (Fig. 9.6).
Providing the inserted gene lacks transcription-
termination signals, transcription of the 3
′
delta
sequence will occur, which is a prerequisite for
transposition. Such constructs act as amplification
cassettes, for, once introduced into yeast, transposi-
tion of the new gene occurs to multiple sites in the
genome (Boeke
et al
. 1988, Jacobs
et al
. 1988).
′
(VLPs), which accumulate in the cytoplasm (Fig. 9.5).
The particles, which have a diameter of 60 - 80 nm,
have reverse-transcriptase activity. The major struc-
tural components of VLPs are proteins produced
by proteolysis of the primary translation product of
