Inverted terminal repeat sequences

In recent years, there have been significant improvements in production and purification of rAAV vectors. The major improvements in production have included enhanced output of the number of DNase resistant particles (drp) per cell and the emergence of scaleable systems. The most widely utilized rAAV vector production methods require four genetic elements (Hermonat and Muzyczka, 1984 Tratschinetal., 1984) (1) mammalian tissue culture cells, (2) vector sequences containing a transgene flanked by AAV inverted terminal repeats, (3) AAV helper sequences comprising... 

Since McClintock s early work on mobile elements in corn, transposons have been identified in other eukaryotes. For Instance, approximately half of all the spontaneous mutations observed in Drosophila are due to the Insertion of mobile elements. Although most of the mobile elements in Drosophila function as retrotransposons, at least one—the P element—functions as a DNA transposon, moving by a cut-and-paste mechanism similar to that used by bacterial insertion sequences. Current methods for constructing transgenic Drosophila depend on engineered, high-level expression of the P-element transposase and use of the P-element Inverted terminal repeats as targets for transposition. 

The core of an adenoviral particle consists of the DNA genome complexed with four polypeptides (pV, pVII, mu, TP). Figure 10.1-4 schematically shows the structure of an adenovirus genome. The extremities contain inverted terminal repeat (ITR) sequences (100-140bp), covalently linked to terminal proteins (TPs), which function as replication origins [23]. The nearby / sequence at the left end of the... 

Figure 10.1-4. The adenovirus genome. The early (E1-E4) and late (L1-L5) transcription units are identified by the arrows. The inverted terminal repeats (ITRs), packaging sequence ( /), and major late promoter (MLP) are also shown. (Adapted from Ref. 26.)...

Ishino, M., Sawada, Y., Yaegashi, T., Demura, M. et al. (1987) Nucleotide sequence of the adenovirus type 40 inverted terminal repeat close relation to that of adenovirus type 5. Virology, 15, 414-416. 

The most essential and unique component in AAV is its inverted terminal repeats (ITRs) at both ends of the viral genome (57). In AAV-2, the ITR is composed of 145 self-complementary nucleotides (Fig. 2). This is an extremely GC (guanosine and cytosine)-rich sequence with a GC content of higher than 80% (the average GC content in human chromosomal DNA is less than 60% (58)). The overall structure ofthe ITR is a T-shaped hairpin with a three-way junction (Fig. 2). The A and A fragments form a duplex stem which terminates with two small palindromes (defined as B, B and C, C arms). Depending on the orientation of these two small palindromes, AAV ITRs can be classified as flip (B arm is closer to the 3 end) or flop (C arm is closer to the 3 end) (29). Biophysical studies have indicated that the ITR is composed of largely... 

Wang XS, Ponnazhagan S, Srivastava A. Rescue and replication of adeno-assoeiated virus type 2 as well as vector DNA sequences from recombinant plasmids containing deletions in the viral inverted terminal repeats selective encapsidation of viral genomes in progeny virions. J Virol 1996 70 1668-1677. 

Wang XS, Qing K, Ponnazhagan S, Srivastava A. Adeno-assoeiated vims type 2 DNA rephcalion in vivo mutation analyses of the D sequence in viral inverted terminal repeats. J Virol 1997 71 3077-3082. 

In other transcriptionally regulated Ad constructs described to date, the El A promoter has been removed and replaced with tumor or tissue-specific promoters (81-84). A list of different promoters that have been used to date is shown in Table 1. In some cases, interference from the nearby inverted terminal repeat (ITR) sequence can result in the loss of promoter strength and fidelity (85). Selectivity can be enhanced with the use of silencer (86) or insulator sequences (87). For example, the AdEla04i viras using the artificial a-fetoprotein (AFP) promoter to express ElA needed six copies of a silencer sequence to repress the promoter activity in normal hepatocytes. This virus showed specificity and potency for hepatoma carcinoma tumors and not in non-AFP- producing cells or in normal hepatocytes (86). 

Characterization of a LINE-like element can be quite difficult. First, the repetitive region of the isolated clone is sequenced. Then a computer analysis is performed to determine the characteristics of the sequence, such as the presence of inverted or direct terminal repeats. If terminal repeats are not found it is still possible that the element is one of the terminal repeat class which has a deletion at one end. Further clones are then obtained from the library by using the repetitive portion of the clone as a probe. By way of cross-hybridization, regions of homology between the two isolates can be delineated. The sequence of portions of other isolates should then be determined to search for the ends of the element. 

In searching for new transposable elements, it is generally not possible to use DNA cross-hybridization to detect related elements from the same class, because the similarity is detectable only in the amino acid sequences. Instead, the features that are diagnostic of some transposable elements are the presence of terminal repeats, whether inverted or direct, and the amino acid sequence of the proteins they encode. Before a new transposable element can be classified, it is necessary to determine the DNA sequence of the element. 

Yes. After the RNA polymerase transcribes the inverted repeat sequence, it is the ability of this region to form a hairpin in the single-strand transcript that is responsible, at least in part, for termination of the chain. 

We infer that it is unlikely that the thermophile terminators resemble bacterial rho-independent terminators. Moreover, this view is reinforced by examining the sequences for the bacterial terminators using a program in the GCG-software package from the University of Wisconsin. This is based on the observations of Brendel and Trifonov [125] that in addition to the inverted repeat preceding a polythymidine sequence many bacterial terminators share sequence features located both upstream and downstream from the termination site. Only one of the termination sites shown in Table 5 is predicted by this program. 

Base sequence of (a) the DNA of the E. coli trp operon at which transcription termination occurs and of (b) the y terminus of the mRNA molecule. The inverted-repeat sequence is indicated by reversed arrows. The mRNA molecule is folded to form a stem-and-loop structure. 

Flanking these structural genes are two direct repeats, the long terminal repeats (LTRs) of about 250 to 1400 base pairs each. Each LTR is flanked in turn by short inverted repeat sequences, 5 to 13 base pairs in length. Integration occurs by a mechanism that duplicates the target site, so that the integrated viral sequence, called a provirus, is flanked by direct repeats of host cell DNA (5 to 13 base pairs each). 

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