SignificanceFixed nitrogen is essential for plant growth. Some plants, such as legumes, can host nitrogen-fixing bacteria within cells in root organs called nodules. Nodules are considered to have evolved in parallel in different lineages, but the genetic changes underlying this evolution remain unknown. Based on gene expression in the nitrogen-fixing nonlegume Parasponia andersonii and the legume Medicago truncatula, we find that nodules in these different lineages may share a single origin. Comparison of the genomes of Parasponia with those of related nonnodulating plants reveals evidence of parallel loss of genes that, in legumes, are essential for nodulation. Taken together, this raises the possibility that nodulation originated only once and was subsequently lost in many descendant lineages.

Middle component RNA (M RNA) of cowpea mosaic virus (CPMV) was transcribed into cDNA and double‐stranded cDNA was inserted into the EcoRI site of plasmid pBRH2. The nucleotide sequence of inserts was determined, after subcloning in bacteriophages M13mp7, M13mp8 or M13mp9, by the dideoxy chain termination method. The complete sequence of CPMV M RNA, up to the poly(A) tail, is 3481 nucleotides long. The sequence contains a long open reading frame starting at nucleotide 161 from the 5′ terminus and continuing to 180 nucleotides from the 3′ terminus. The sequence does not contain a polyadenylation signal for the poly(A) tail at the 3′ end of CPMV RNA. The initiation site at position 161 together with AUG codons in the same reading frame at positions 512 and/or 524 account for the two large colinear precursor polypeptides translated in vitro from M RNA. The amino acid sequence deduced from the nucleotide sequence suggests that both precursor polypeptides are proteolytically cleaved at glutaminyl‐methionine and glutaminyl‐glycine, respectively, to produce the two viral capsid proteins.

The major coat protein of the filamentous bacteriophage M13 is located in the inner membrane of host cell Escherichia coli prior to assembly into virions. To identify the transmembrane domain of the coat protein, we have introduced unique cysteine residues along the putative transmembrane domain at position 25, 31, 33, 36, 38, 46, 47, 49, or 50. The mutant major coat protein was solubilized by membrane-mimicking detergents or reconstituted into mixed bilayers of phospholipids. Information about the environmental polarity was deduced from the wavelength of maximum emission, using N-[[(iodoacetyl)-amino)ethyl]-1-sulfonaphthylamine (IAEDANS) attached to the SH groups of the cysteines as a fluorescent probe. Additional information was obtained by determining the accessibility of AEDANS for the fluorescence quencher molecules acrylamide and 5-doxylstearic acid, and the reactivity of the cysteine's sulfhydryl group toward 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). Our data suggest transmembrane boundaries close to residue 25 and 46, with residue 25 inside the hydrophobic part of the membrane in very close proximity to the membrane-water interface and residue 46 located at the membrane-water interface. Domains of the mutant coat protein which are packed or coated by cholate molecules and various other detergents [except for sodium dodecyl sulfate (SDS)] are at least similarly packed by phospholipid molecules in bilayers. SDS is a good solubilizing detergent but badly mimics the typical nature of a membrane structure. The overall results are interpreted with respect to the established conformation of the coat protein and its membrane anchoring mechanism.

A series of specific deletion mutants derived from a full-length cDNA clone of cowpea mosaic virus (CPMV) B RNA was constructed with the aim to study the role of viral proteins in the proteolytic processing of the primary translation products. For the same purpose cDNA clones were constructed having sequences derived from both M and B RNA of CPMV. In vitro transcripts prepared from these clones with T7 RNA polymerase, were efficiently translated in rabbit reticulocyte lysates. The translation products obtained were processed in the lysate by specific proteolytic cleavages into smaller products, which made it possible to study subsequently the effect of the various mutations on this process. The results obtained indicate that the B RNA-encoded 24K polypeptide represents a protease responsible for all cleavages in the polyproteins produced by both CPMV B and M RNA. For efficient cleavage of the glutamine-methionine site in the M RNA encoded polyprotein the presence of a second B RNA encoded protein, the 32K polypeptide, is essential, although the 32K polypeptide itself does not have proteolytic activity. A number of cleavage-site mutants were constructed in which the coding sequence for the glutamine-glycine cleavage site between the two capsid proteins was changed. Subsequent in vitro transcription and translation of these cleavage site mutants show that a correct dipeptide sequence is a prerequisite for efficient cleavage but that the folding of the polypeptide chain also plays an important role in the formation of a cleavage site.

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