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M Frilander

Publications and source records attributed to M Frilander.

13 recordsLinked to original sources
PubMed1995

Differentiation between minus- and plus-strand synthesis: polymerase activity of dsRNA bacteriophage phi 6 in an in vitro packaging and replication system.

Empty procapsids of the segmented dsRNA virus phi 6, produced in Escherichia coli from a cloned L genome segment, package plus-strand phi 6 ssRNA genomic segments, synthesize minus strands, and transcribe the newly formed dsRNA templates. Procapsids can be restricted to minus-strand synthesis by high concentrations of CaCl2 or low concentrations of nucleotides, enabling us to separate the viral minus-strand (replication) and plus-strand (transcription) RNA-dependent RNA polymerase activities in vitro. Reaction conditions for minus-strand synthesis were optimized. Plus-strand synthesis by procapsids could be activated by binding of purine nucleoside triphosphates to a low-affinity NTP-binding site. The second 5'-terminal nucleotide of the phi 6 plus-sense ssRNA L genomic segment is important for determining the level of transcription of that segment and the generation of infectious procapsids.

Bacteriophage phi 6↗
PubMed1995

In vitro packaging of the single-stranded RNA genomic precursors of the segmented double-stranded RNA bacteriophage phi 6: the three segments modulate each other's packaging efficiency.

Bacteriophage phi 6 is a double-stranded RNA (dsRNA) virus that has a genome composed of three linear dsRNA segments (l, m, s). These are encapsidated into a dodecahedral procapsid particle consisting of proteins P1, P2, P4 and P7. Expression of the cDNA copy of the L segment in Escherichia coli leads to the formation of empty procapsid particles. These particles are able to package the plus-sense single-stranded RNA (ssRNA)s of each genome segment in vitro. We have used this in vitro system for a detailed study of phi 6 RNA packaging. The reaction conditions for RNA packaging were optimized using a RNase protection assay. The RNA packaging reaction is dependent on divalent cations (either Mg2+ or Mn2+) and requires a nucleoside triphosphate (NTP) as an energy source. Any one of the rNTPs, dNTPs or ddNTPs can support the RNA packaging. Purine nucleotides support packaging better than pyrimidine nucleotides, GTP being preferred to ATP. The plus-sense ssRNA of each the three genome segments can be packaged independently into the procapsid. However, when two or three segments are packaged simultaneously, regulatory effects modulating the packaging efficiency can be detected between the segments. The packaging of the s and m segments is more efficient when they are packaged alone, compared to a situation in which they are packaged with the other segments. In contrast, the packaging of the l segment is very inefficient alone, but is enhanced when packaged together with the m segment. We propose that each segment has a preferred high-affinity binding site in the procapsid particle and packaging of the m segment creates the high-affinity binding site for the l segment. If any of the segments is missing from the packaging reaction the other segments can occupy its binding site.

Bacteriophage phi 6↗
PubMed1995

The large genome segment of dsRNA bacteriophage phi6 is the key regulator in the in vitro minus and plus strand synthesis.

Bacteriophage phi6 is a double-stranded RNA (dsRNA) virus that has a genome composed of three linear dsRNA segments (L, M, S). These are encapsidated into a dodecahedral procapsid particle consisting of proteins P1, P2, P4, and P7. Empty preformed procapsids are able to package the plus-sense single-stranded RNA (ssRNA) of each genome segment, to synthesize the corresponding minus strands ("replication") to form dsRNA segments, and to continue to the plus strand synthesis ("transcription") in which the dsRNA segments are used as templates in production of plus-sense ssRNA. In this study, we have investigated the requirements for the switch-on of minus and plus strand syntheses. We show that there exists an inverse relationship between regulation of the ssRNA packaging and minus strand synthesis. The packaging of single-stranded l, which has previously been shown to be packaged as the last, is the necessary signal for the onset of the minus strand synthesis. The absolute requirement for plus strand synthesis is minus strand synthesis of l, but in addition, the minus strand synthesis of m and the packaging of s segment are needed for efficient plus strand synthesis. Furthermore, the second nucleotide at the 5'-end of each segment regulates the extent of the transcription.

Bacteriophage phi 6↗
PubMed1994

Identification of the packaging regions within the genomic RNA segments of bacteriophage phi 6.

Bacteriophage phi 6 has a genome of three segments of double-stranded RNA enclosed in a procapsid composed of four different proteins. The preformed procapsid is capable of packaging plus-strand transcripts of the genomic segments in an in vitro reaction. The packaging-specific sequences on the RNA molecules are located near the 5' ends. In this study we show that the packaging sequences are different for each of the three segments and that they are of about 250 nucleotides in length. Although these sequences are consistent with some secondary structure, there is no clear structural similarity between the packaging regions of the three segments.

Bacteriophage phi 6↗
PubMed1994

RNA structural requirements for stability and minus-strand synthesis in the dsRNA bacteriophage phi 6.

Bacteriophage phi 6 has a genome consisting of three segments of double-stranded RNA designated L, M, and S. Each virion contains one of each genomic segment. Empty procapsids can package plus-strand transcripts of the genomic segments if the 5' regions are intact. Minus-strand synthesis takes place if all three segments are packaged and if the 3' end of the segment is intact. The 3' ends of the segments contain four hairpin structures within a region of high sequence conservation. We now show that removal of parts of this region leads to progressive but limited loss of ability to support minus-strand synthesis. The defective 3' ends can be corrected by heterologous recombination with the termini of other segments. Segments that have small deletions in the conserved region and that support apparently normal minus-strand synthesis are highly recombinogenic.

Bacteriophage phi 6↗
PubMed1993

RNA structure and heterologous recombination in the double-stranded RNA bacteriophage phi 6.

Bacteriophage phi 6 has a genome of three segments of double-stranded RNA, designated L, M, and S. A 1.2-kbp kanamycin resistance gene was inserted into segment M but was shown to be genetically unstable because of a high recombination rate between segment M and the 3' ends of segments S and L. The high rate of recombination is due to complementary homopolymer tracts bounding the kan gene. Removal of one arm of this potential hairpin stabilizes the insertion. The insertion of a 241- or 427-bp lacZ' gene into segment M leads to a stable Lac+ phage. The insertion of the same genes bounded by complementary homopolymer arms leads to recombinational instability. A stable derivative of this phage was shown to have lost one of the homopolymer arms. Several other conditions foster recombination. The truncation of a genomic segment at the 3' end prevents replication, but such a damaged molecule can be rescued by recombination. Similarly, insertion of the entire 3-kb lacZ gene prevents normal formation of virus, but the viral genes can be rescued by recombination. It appears that conditions leading to the retardation or absence of replication of a particular genomic segment facilitate recombinational rescue.

Bacteriophage T7↗
PubMed1992

In vitro packaging and replication of individual genomic segments of bacteriophage phi 6 RNA.

The genome of bacteriophage phi 6 contains three segments of double-stranded RNA. Procapsid structures whose formation was directed by cDNA copies of the large genomic segment are capable of packaging the three viral message sense RNAs in the presence of ATP. Addition of UTP, CTP, and GTP results in the synthesis of minus strands to form double-stranded RNA. In this report, we show that procapsids are capable of taking up any of the three plus-strand single-stranded RNA segments independently of the others. In manganese-containing buffers, synthesis of the corresponding minus strand takes place. In magnesium-containing buffers, individual message sense viral RNA segments were packaged, but minus-strand replication did not take place unless all three viral single-stranded RNA segments were packaged. Since the conditions of packaging in magnesium buffer more closely resemble those in vivo, these results indicated that there is no specific order or dependence in packaging and that replication is regulated so that it does not begin until all segments are in place.

Capsid↗
PubMed1992

Dependence of minus-strand synthesis on complete genomic packaging in the double-stranded RNA bacteriophage phi 6.

Bacteriophage phi 6 has a segmented genome consisting of three pieces of double-stranded RNA (dsRNA). The viral procapsid is the structure that packages plus strands, synthesizes the complementary negative strands to form dsRNA, and then transcribes dsRNA to form plus-strand message. The minus-strand synthesis of a particular genomic segment is dependent on prior packaging of the other segments. The 5' end of the plus strand is necessary and sufficient for packaging, while the normal 3' end is necessary for synthesis of the negative strand. We have now investigated the ability of truncated RNA segments which lack the normal 3' end of the molecules to stimulate the synthesis of minus strands of the other segments. Fragments missing the normal 3' ends were able to stimulate the minus-strand synthesis of intact heterologous segments. Minus-strand synthesis of one intact segment could be stimulated by the presence of two truncated nonreplicating segments. The 5' fragments of each single-stranded genomic segment can compete with homologous full-length single-stranded genomic segments in minus-strand synthesis reactions, suggesting that there is a specific binding site in the procapsid for each segment.

Bacteriophages↗
PubMed1991

Genome organization of membrane-containing bacteriophage PRD1.

We have determined the nucleotide sequence of the late region (11 kbp) of the lipid-containing bacteriophage PRD1. Gene localization was carried out by complementing nonsense phage mutants with genomic clones containing specific reading frames. The localization was confirmed by sequencing the N-termini of isolated gene products as well as sequencing the N-termini of tryptic fragments of the phage membrane-associated proteins. This, with the previously obtained sequence of the early regions, allowed us to organize most of the phage genes in the phage genome.

Amino Acid Sequence↗