Comparative genomics shows that a substantial fraction of the genes in sequenced genomes encodes ‘conserved hypothetical’ proteins, i.e. those that are found in organisms from several phylogenetic lineages but have not been functionally characterized. Here, we briefly discuss recent progress in functional characterization of prokaryotic ‘conserved hypothetical’ proteins and the possible criteria for prioritizing targets for experimental study. Based on these criteria, the chief one being wide phyletic spread, we offer two ‘top 10’ lists of highly attractive targets. The first list consists of proteins for which biochemical activity could be predicted with reasonable confidence but the biological function was predicted only in general terms, if at all (‘known unknowns’). The second list includes proteins for which there is no prediction of biochemical activity, even if, for some, general biological clues exist (‘unknown unknowns’). The experimental characterization of these and other ‘conserved hypothetical’ proteins is expected to reveal new, crucial aspects of microbial biology and could also lead to better functional prediction for medically relevant human homologs.

/pdf/conserved-hypothetical-proteins-prioritization-of-targets-3r44nfz6n2.pdf

‘Conserved hypothetical’ proteins: prioritization of targets for experimental study

The immutans (im) mutant of Arabidopsis shows a variegated phenotype comprising albino and green somatic sectors. We have cloned the IM gene by transposon tagging and show that even stable null alleles give rise to a variegated phenotype. The gene product has amino acid similarity to the mitochondrial alternative oxidase. We show that the IM protein is synthesized as a precursor polypeptide that is imported into chloroplasts and inserted into the thylakoid membrane. The albino sectors of im plants contain reduced levels of carotenoids and increased levels of the carotenoid precursor phytoene. The data presented here are consistent with a role for the IM protein as a cofactor for carotenoid desaturation. The suggested terminal oxidase function of IM appears to be essential to prevent photooxidative damage during early steps of chloroplast formation. We propose a model in which IM function is linked to phytoene desaturation and, possibly, to the respiratory activity of the chloroplast.

/pdf/mutations-in-the-arabidopsis-gene-immutans-cause-a-3y6fov8k37.pdf

Mutations in the Arabidopsis gene IMMUTANS cause a variegated phenotype by inactivating a chloroplast terminal oxidase associated with phytoene desaturation.

https://archive-ouverte.unige.ch/unige:126540/ATTACHMENT01

Participation of nuclear genes in chloroplast gene expression.

Nuclear gene-induced variegation mutants provide a powerful system to dissect interactions between the genetic systems of the nucleus-cytoplasm, the chloroplast, and the mitochondrion. The immutans (im) variegation mutation of Arabidopsis is nuclear and recessive and results in the production of green- and white-sectored leaves. The green sectors contain cells with normal chloroplasts, whereas the white sectors are heteroplastidic and contain cells with abnormal, pigment-deficient plastids as well as some normal chloroplasts. White sector formation can be promoted by enhanced light intensities, but sectoring becomes irreversible early in leaf development. The white sectors accumulate the carotenoid precursor phytoene. We have positionally cloned IM and found that the gene encodes a 40.5-kD protein with sequence motifs characteristic of alternative oxidase, a mitochondrial protein that functions as a terminal oxidase in the respiratory chains of all plants. However, phylogenetic analyses revealed that the IM protein is only distantly related to these other alternative oxidases, suggesting that IM is a novel member of this protein class. We sequenced three alleles of im, and all are predicted to be null. Our data suggest a model of variegation in which the IM protein functions early in chloroplast biogenesis as a component of a redox chain responsible for phytoene desaturation but that a redundant electron transfer function is capable of compensating for IM activity in some plastids and cells.

/pdf/the-immutans-variegation-locus-of-arabidopsis-defines-a-47jykomq62.pdf

The IMMUTANS variegation locus of Arabidopsis defines a mitochondrial alternative oxidase homolog that functions during early chloroplast biogenesis.

The pentatricopeptide repeat (PPR) is a degenerate 35–amino acid repeat motif that is widely distributed among eukaryotes. Genetic, biochemical, and bioinformatic data suggest that many PPR proteins influence specific posttranscriptional steps in mitochondrial or chloroplast gene expression and that they may typically bind RNA. However, biological functions have been determined for only a few PPR proteins, and with few exceptions, substrate RNAs are unknown. To gain insight into the functions and substrates of the PPR protein family, we characterized the maize (Zea mays) nuclear gene ppr4, which encodes a chloroplast-targeted protein harboring both a PPR tract and an RNA recognition motif. Microarray analysis of RNA that coimmunoprecipitates with PPR4 showed that PPR4 is associated in vivo with the first intron of the plastid rps12 pre-mRNA, a group II intron that is transcribed in segments and spliced in trans. ppr4 mutants were recovered through a reverse-genetic screen and shown to be defective for rps12 trans-splicing. The observations that PPR4 is associated in vivo with rps12-intron 1 and that it is also required for its splicing demonstrate that PPR4 is an rps12 trans-splicing factor. These findings add trans-splicing to the list of RNA-related functions associated with PPR proteins and suggest that plastid group II trans-splicing is performed by different machineries in vascular plants and algae.

/pdf/a-pentatricopeptide-repeat-protein-facilitates-the-trans-1djguoforl.pdf

A Pentatricopeptide Repeat Protein Facilitates the trans-Splicing of the Maize Chloroplast rps12 Pre-mRNA

Iojap (ij) is a recessive striped mutant of maize affecting the development of plastids in a local and position-dependent manner on the leaves. The ij-affected plastids are transmitted to some of the progeny even when the function of the nuclear gene is restored. Developmental defects during embryogenesis and leaf proliferation are other phenotypic characteristics of ij. The extent of striping and the degree of developmental arrest in ij depend upon genetic background. To understand the diverse and unique phenotypic expression of ij, a transposon tagging experiment has been conducted using Robertson's Mutator (Mu). A new ij mutant was obtained from crosses of the reference allele of (ij-ref) to Mu lines. Subsequent genetic and molecular studies showed that the mutant carried a new ij allele (ij-mum1) from the Mu lines and contained a Mu1 element that cosegregated with the iojap phenotype. A 6.0 kb EcoRI genomic DNA fragment containing the Mu1 element was cloned. ij-ref is unstable, and revertants (Ij-Rev) have been obtained. Using the flanking DNA from the genomic clone as a probe, DNA polymorphisms were detected between ij-ref and these revertants. Further, transcripts were restored to the normal level in Ij-Rev seedlings. Comparison of genomic DNA clones from ij-ref, ij-mum1 and Ij indicated that the ij-ref allele contained 1.5 kb of additional DNA related to a transposable element, Ds. Germinal and somatic revertant alleles were derived by excision of this 1.5 kb element from ij-ref. The structure of the Ij gene and the DNA sequence of its transcribed region were determined. The Ij gene encodes a 24.8 kDa protein that showed no significant sequence similarity with proteins listed in databases.

https://www.embopress.org/doi/epdf/10.1002/j.1460-2075.1992.tb05497.x

C.D. Han

Papers

Molecular cloning and characterization of iojap (ij), a pattern striping gene of maize.