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K I Berns

Publications and source records attributed to K I Berns.

At least 73 records · Page 4Linked to original sources
PubMed1984

Unique events in parvovirus replication.

The single-stranded parvovirus genome is characterized by a palindromic sequence at the 3' terminus. This structure serves uniquely as a primer for DNA replication.

Base Sequence↗
PubMed1983

Rescue of adeno-associated virus from recombinant plasmids: gene correction within the terminal repeats of AAV.

We have isolated three types of pBR322-AAV recombinant plasmids that contain deletions within the 145 bp AAV terminal repeats. When the plasmids were transfected into human cells, mutants that contained deletions within the left (type I) or right (type II) terminal repeat were viable. Of four mutants examined that contained deletions in both termini (type III), only one was viable. All of the viable mutants produced AAV virions that contained wild-type AAV DNA. Furthermore, the viable type III deletion could be converted to a nonviable mutant by deleting all copies of an 11 bp sequence from its termini. We conclude that there is an efficient mechanism for correcting deletions within the AAV termini. A model that could account for these observations is also discussed.

Chromosome Deletion↗
PubMed1983

Nucleotide sequence and organization of the adeno-associated virus 2 genome.

The complete nucleotide sequence of the adeno-associated virus 2 genome was determined. The single-stranded genome is 4,675 nucleotides in length and contains inverted terminal repeats of 145 nucleotides, the first 125 nucleotides of which form a palindromic sequence. Within the inverted terminal repetitions, there are two distinct sequences representing an inversion of 43 nucleotides that can exist on either terminus. The 5' and 3' termini of three major mRNA transcripts, which are present in both spliced and unspliced forms, were also mapped on the viral genome. Potential initiation and termination codons for efficient protein synthesis were identified, and genome segments were assigned that code for three major viral capsid proteins and, possibly, some as-yet-unidentified, nonstructural viral proteins.

Amino Acid Sequence↗
PubMed1982

Polyoma-like particle: characterization of the DNA encapsidated in vitro by polyoma empty capsids.

Polyoma empty capsids and DNA interact in a cell-free system to form a polyoma-like particle (PLP). The particle is stable in high concentrations of salt and contains a DNA fragment that is protected by the capsid from the action of DNase I. The DNA extracted from PLP is heterogeneous in size. It has a mean molecular weight of 1.2 x 10(6) with a standard deviation of +/- 0.5 x 10(6). Analysis of PLP DNA with restriction endonucleases and the Southern blot technique reveals that a specific primary sequence is not required for PLP formation. Either linear, circular, or supercoiled polyoma DNA, as well as single-stranded DNA, rRNA, and the synthetic homopolymers poly(dA) . poly(dT) and poly(dG) . poly(dC) can be used for PLP formation. It is concluded that polyoma empty capsids can be used to bind and encapsidate nucleic acids differing in their primary, secondary, and superhelical tertiary structure to form, in vitro, stable polyoma-like particles.

Capsid↗
PubMed1982

Cloning of adeno-associated virus into pBR322: rescue of intact virus from the recombinant plasmid in human cells.

We have cloned intact duplex adeno-associated virus (AAV) DNA into the bacterial plasmid pBR322. The AAV genome could be rescued from the recombinant plasmid by transfection of the plasmid DNA into human cells with adenovirus 5 as helper. The efficiency of rescue from the plasmid was sufficiently high to produce yields of AAV DNA comparable to those observed after transfection with equal amounts of purified virion DNA. Thus, the recombinant plasmid itself may be a model for studying the rescue of a latent AAV viral infection. In addition, the efficient rescue of viable AAV from the recombinant plasmid should facilitate the genetic analysis of AAV. Finally, the results of an analysis of the DNA from rescued virions indicate that an inversion of the AAV terminal sequences occurred during replication.

Cells, Cultured↗
PubMed1981

Inverted terminal repetition in adeno-associated virus DNA: independence of the orientation at either end of the genome.

Complementary strands of adeno-associated virus DNA labeled with 32P at the 5' ends were separated and then self-annealed to form single-stranded circles stabilized by hydrogen bonds between the complementary sequences in the inverted terminal repetitions. We have previously shown that there are two distinct sequences in the terminal repetition which represent an inversion of the first 125 nucleotides (E. Lusby et al., J. Virol. 34:402-409, 1980; I. S. Spear et al., Virology 24:627-634, 1977). Base pairing between terminal sequences of the same orientation leads to a normal double helical structure. If sequences of the opposite orientation pair, an aberrant secondary structure is formed. HpaII digestion of the self-annealed, single-stranded circles led to labeled terminal fragments that corresponded both to those generated from termini of a normal double helical structure and those generated from an aberrant terminal secondary structure. Thus, the orientation of the terminal repetition at one end of the genome is not influenced by the orientation at the other end.

DNA Replication↗
PubMed1980

Integration of the adeno-associated virus genome into cellular DNA in latently infected human Detroit 6 cells.

A clone of human cells (Detroit 6) latently infected by adeno-associated virus (AAV) has been characterized with regard to the status of the viral DNA. In both early (9 to 10) and late (118) passages of the clone, AAV-DNA was recombined with host DNA, at least in some cases as a head-to-tail tandem repeat, via the terminal sequences of the viral genome. However, it was not possible to distinguish between integration into chromosomal DNA and very large plasmids (< 20 x 10(6) molecular weight) which contain both viral and cellular DNA sequences. Although evidence for some modifications of the viral sequence was obtained, most of the integrated sequences appeared to be intact. In some cases sequences of undetermined origin separated adjacent copies of the viral genome. Free copies of the AAV genome were detectable in late passage cells, but not in early passage cells. The orientation of nucleotide sequences present in the free AAV DNA from late passage cells was indistinguishable from that of virion DNA. With the notable exception, the organization of the integrated AAV sequences as determined by restriction enzyme digestion remained constant with continued passage. Digestion with SmaI, which cleaves within the palindromic region of the terminal repetition in AAV DNA, produced reproducibly different patterns when early and late passage DNAs were compared. Several models for rescue of free copies of the genome from the integrated DNA are possible, all of which involve the terminal repetition.

Base Sequence↗
PubMed1980

Nucleotide sequence of the inverted terminal repetition in adeno-associated virus DNA.

The inverted terminal repetition in adeno-associated virus type 2 DNA has been sequenced. The terminal repetition contain 145 nucleotides of which the first 125 nucleotides can self-base pair to form a T-shaped hairpin structure. Both restriction endonuclease analysis with SmaI and BglI and direct sequence analysis of the SmaI fragments provide evidence for two sequences in the region of the terminal repetition between nucleotides 44 and 81. The two sequences represent an inversion of the first 125 nucleotides of the terminal repetition. Based on these data a model for adeno-associated virus DNA replication is presented which agrees in detail with a general model for eucaryotic DNA replication originally proposed by Cavalier-Smith (T. Cavalier-Smith, Nature [London] 18:672--684, 1976).

Base Sequence↗
PubMed1977

Evidence for two nucleotide sequence orientations within the terminal repetition of adeno-associated virus DNA.

Duplex adeno-associated virus (AAV) DNA, produced by annealing plus and minus virion single strands, has been digested with several bacterial restriction endonucleases. These studies reveal the existence of alternate secondary structures at the termini of duplex AAV DNA. Analysis of the sites of endo R-Hpa II cleavage, the products of complete endo R-Hpa II digestion, and the multiple terminal secondary structures leads to the conclusion that there are two possible nucleotide sequences at each end of AAV DNA. A model that attributes the terminal nucleotide sequence heterogeneity to two possible orientations of the first 120 nucleotides at each end of the DNA is proposed; in one case the sequence is 1 to 120; in the other case the sequence is inverted. An origin of the inversion is suggested based on previously described intermediates in AAV DNA replication.

Base Sequence↗
PubMed1976

Genome localization of adeno-associated virus RNA.

In previous work, linear duplex molecules of adeno-associated virus, type 2 (AAV2), DNA were cleaved with the restriction endonucleases R-EcoRI, R-HindII, and R-HindIII. The physical order of the specific fragments obtained was deduced and oriented with respect to the DNA strand polarity and the direction of transcription. Stable AAV RNA is transcribed only from 70% of the minus DNA strand. We report here RNA-DNA hybridization experiments using these restriction fragments to obtain a more accurate map of the portion of the AAV genome represented in stable RNA. The data obtained with several sets of restriction fragments annealed to either whole-cell RNA or poly(A)-containing RNA were internally consistent. The AAV RNA annealed with a continuous region of the AAV DNA, beginning at 0.18 map units (18%) from the left end of the molecule and ending at 0.88 map units. In addition, the restriction endonuclease BamHI was found to make one specific cleavage in AAV2 DNA at 0.22 map units, which is 0.04 map units (i.e., 160 nucleotides) to the right (""down stream'') of the point corresponding to the 5' end of the viral mRNA.

DNA Restriction Enzymes↗