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RNA-seq is commonly used to identify genetic modules that respond to perturbations. In single cells, transcriptomes have been used as phenotypes, but this concept has not been applied to whole-organism RNA-seq. Linear models can quantify expression effects of individual mutants and identify epistatic effects in double mutants. To make interpretation of these high-dimensional measurements intuitive, we developed a single coefficient to quantify transcriptome-wide epistasis that accurately reflects the underlying interactions. To demonstrate our approach, we sequenced four single and two double mutants ofCaenorhabditis elegans. From these mutants, we reconstructed the known hypoxia pathway. In addition, we uncovered a class of 56 genes that have opposing changes in expression inegl-9(lf)compared tovhl-1(lf)but theegl-9(lf); vhl-1(lf)mutant has the same phenotype asegl-9(lf). This class violates the classical model of HIF-1 regulation, but can be explained by postulating a role of hydroxylated HIF-1 in transcriptional control.Significance StatementTranscriptome profiling is a way to quickly and quantitatively measure gene expression level. Because of their quantitative nature, there is widespread interest in using transcriptomic profiles as a phenotype for genetic analysis. However, a source of major concern is that whole-animal transcriptomic profiles mix the expression signatures of multiple cellular states, making it hard to accurately reconstruct genetic interactions. Additionally, it has been difficult to quantify epistasis, the signature of genetic interaction between two genes, in these molecular phenotypes. Here, we show that it is possible to accurately reconstruct genetic interactions between genes using whole-animal RNA sequencing, and we demonstrate a powerful new way to measure and understand epistasis arising from these measurements. This suggests that whole-organism RNA-seq can be a powerful tool with which to understand genetic interactions in entire organisms and not only in isolated cells. With the advent of genome engineering tools, generating mutants has become easier and faster for many organisms. As mutants become easier to create, phenotyping them has become a major bottleneck in understanding the biological functions of the genes in question. Our work presents a possible solution to this problem, because transcriptome profiling is fast and sensitive to genetic perturbations regardless of the context they operate in.

We describe a method for identifying phosphoproteins among total cellular proteins using two-dimensional electrophoresis (2DE). The method involves the simultaneous double-labeling of cells with [ 35 S]methionine and [ 32 P]orthophosphate. Proteins that incorporate one or both isotopes are then identified within the same gel on the basis of the different energies of the respective isotope emissions. Detection is achieved using a storage phosphor screen that is exposed to the dried 2DE gel with or without (+/−) the insertion of a copper filter between the gel and the storage phosphor screen. Each of these two accumulated potential images is digitized on a Phosphorimager. Because both +/− copper-filtered images are derived from the same gel, it is possible to precisely overlay the two images. Measuring the efficiency of transfer of radiation across the copper for each spot allows accurate determination of whether there is a double or single label in each protein. The dynamic range of the method allows quantitation over five orders of magnitude for a single isotope and over four orders of magnitude for 32 P and 35 S isotope mixtures in the 2DE gel. The sensitivity is more than 10-fold greater than that of autoradiography and requires exposures of 2–48 h instead of several weeks. We describe the application of this method to the identification of protein phosphorylation events in a rodent cell line (PC12 cells) after exposure to nerve growth factor. This method should have wide applicability in identifying post-translational modifications of proteins involved in many biological processes.

Glutamate receptors are the predominant excitatory neurotransmitter receptors in the mammalian brain and are classified on the basis of their activation by different agonists. The agonists kainate and alpha-amino-3-hydroxy-5-methyl-4-isoxasolepropionic acid define a class of glutamate receptors termed kainate receptors. We have isolated and sequenced a human glutamate receptor (GluHI) cDNA and determined the chromosomal localization of its gene. The DNA sequence of GluHI would encode a 907-amino acid protein that has a 97% identity to one of the rodent kainate receptor subunits. Many of the changes between the predicted amino acid sequence of GluHI and the most similar rodent kainate receptor (GluRI) occur in a region of the protein encoded in rodents by an alternatively spliced exon. The extreme conservation between the human and rat kainate receptor subunits suggests that a similar gene family will encode human kainate receptors. The GluHI mRNA is widely expressed in human brain. The human gene encoding the GluHI subunit is located at 5q33. While the GluHI gene is not located near a chromosomal region associated with any human neurogenetic disorders, the homologous region on mouse chromosome 11 contains the sites of five neurologic mutations.

Myelin is a highly specialized membrane unique to the nervous system that ensheaths axons to permit the rapid saltatory conduction of impulses. The elaboration of a compact myelin sheath is disrupted in a diverse spectrum of human disorders, many of which are of unknown etiology. The X chromosome-linked human disorder Pelizaeus-Merzbacher disease is a clinically and pathologically heterogeneous group of disorders that demonstrate a striking failure of oligodendrocyte differentiation. This disease appears pathologically and genetically to be similar to the disorder seen in the dysmyelinating mouse mutant jimpy, which has a point mutation in the gene encoding an abundant myelin protein, proteolipid protein (PLP). We report that the molecular defect in one Pelizaeus-Merzbacher family is likewise a point mutation in the PLP gene. A single T----C transition results in the substitution of a charged amino acid residue, arginine, for tryptophan in one of the four extremely hydrophobic domains of the PLP protein. The identification of a mutation in this Pelizaeus-Merzbacher family should facilitate the molecular classification and diagnosis of these X chromosome-linked human dysmyelinating disorders.

Mice homozygous for the myelin-deficient (mld) mutation have an unusual phenotype in which the gene encoding myelin basic protein (MBP) is expressed at low levels and on an abnormal developmental schedule. In this report we describe the organization of the mld MBP locus, which results in this alteration of MBP expression. The mld MBP locus consists of two tandem MBP genes, with the upstream gene containing an inversion of its 3' region. We also demonstrate that although there are low steady-state levels of MBP RNA in mld mice, the mld MBP locus is transcribed at a rate comparable to that of the wild-type MBP gene, indicating that the MBP transcripts are abnormally unstable.

We have isolated cDNA clones encoding the four different forms of mouse myelin basic protein (MBP) and have analyzed the structure of the MBP gene. The three larger forms of MBP differ from the smallest by the inclusion of either or both of two short amino acid sequences at positions 57 and 124 of the smallest protein. The mouse genome contains a single MBP gene comprised of seven exons. The two amino acid sequences present only in the larger MBPs are encoded by separate exons. Furthermore, all exons in the coding region begin or end in complete codons so that alternative splicing does not alter the reading frame. We conclude that the four forms of this myelin protein are encoded in separate mRNAs, each derived by a simple alternative splicing of the primary MBP gene transcript. Comparison of the amino acid sequence encoded by each exon with a recent model of the secondary structure of MBP suggests that each of the seven exons encodes one or two of the predicted structural motifs of the protein.

Mice homozygous for the mutation myelin deficient ( mld ), an allele of shiverer , exhibit decreased CNS myelination, tremors, and convulsions of progressively increasing severity leading to an early death. In this report we demonstrate in mid mice that the gene encoding myelin basic protein (MBP) is expressed at decreased levels and on an abnormal temporal schedule relative to the wild-type gene. Southern blot analyses, field-inversion gel electrophoresis studies, and analyses of mld MBP cosmid clones indicate that there are multiple linked copies of the MBP gene in mld mice. We have introduced an MBP transgene into mld mice and found that myelination increases and tremors and convulsions decrease. Mld and shiverer mice with zero, one, or two copies of the MBP transgene express distinct levels of MBP mRNA and myelin. The availability of a range of mice expressing graded levels of myelin should facilitate quantitative analysis of the roles of MBP in the myelination process and of myelin in nerve function.

cDNA clones encoding proteolipid protein (PLP) were isolated from a mouse brain library and sequenced. We describe two transcripts arising from the PLP locus by alternative splicing: the major one encodes the 277-amino acid PLP protein and the minor one corresponds to the DM-20 protein, a PLP-like protein of 20,000 Mr that shares both amino and carboxyl regions with PLP. These two transcripts lack approximately 70 bases in PLP mRNA from the dysmyelinating jimpy mutant. The deletion spans amino acids 208-232; however, this region is present in the jimpy PLP-encoding gene. We propose that the jimpy mutant suffers a point mutation or the deletion of a few bases in the PLP gene that alters the normal splicing pattern and generates partially deleted PLP transcripts.

Antiserum to a multisubunit DNA-dependent RNA polymerase from vaccinia virions was prepared to carry out genetic studies. This antiserum selectively inhibited the activity of the viral polymerase but had no effect on calf thymus RNA polymerase II. The specificity of the antiserum was further demonstrated by immunoprecipitation of RNA polymerase subunits from dissociated virus particles. The presence in vaccinia virus-infected cells of mRNA that encodes the polymerase subunits was determined by in vitro translation. Immunoprecipitable polypeptides with Mrs of about 135,000, 128,000, 36,000, 34,000, 31,000, 23,000, 21,000, 20,000, and 17,000 were made when early mRNA was added to reticulocyte extracts. The subunits were encoded within the vaccinia virus genome, as demonstrated by translation of early mRNA that hybridized to vaccinia virus DNA. The locations of the subunit genes were determined initially by hybridization of RNA to a series of overlapping 40-kilobase-pair DNA fragments that were cloned in a cosmid vector. Further mapping was achieved with cloned HindIII restriction fragments. Results of these studies indicated that RNA polymerase subunit genes are transcribed early in infection and are distributed within the highly conserved central portion of the poxvirus genome in HindIII fragments E, J, H, D, and A.

We have isolated cDNA clones encoding three separate forms of human myelin basic protein (MBP), 21.5, 18.5, and 17.2 kDa, and have determined the nucleotide sequence of each. The three forms share a common sequence but differ by the inclusion of a 26-residue amino acid sequence near the N terminus of the 21.5-kDa protein or by the absence of an 11-residue amino acid sequence near the C terminus of the 17.2-kDa protein. The sequences either added to or deleted from the major 18.5-kDa MBP correspond exactly to exons 2 and 5 of the mouse MBP gene, suggesting that the human and mouse genes have similar exon structures. We have also identified the 21.5-kDa human MBP on immunoblots using antisera raised to a peptide encoded by the mouse exon 2 sequence. Southern blotting studies of human genomic DNA reveal a simple pattern consistent with a single human MBP gene. Thus, the three MBP mRNAs are likely to arise from alternative splicing of a primary human MBP transcript. Conservation of the 26 amino acid mouse exon 2 sequence in human MBP suggests an important role for this sequence in myelination.

A soluble system that specifically and accurately initiates transcription on defined vaccinia virus templates has been obtained from lysates of infected cells. The required regulatory signals are contained within a DNA segment extending about 230 bp upstream and 30 bp downstream of the RNA start site. Transcription is resistant to alpha-amanitin and inhibited by antibodies to the viral RNA polymerase. Whole cell extracts from uninfected cells cannot accurately transcribe vaccinia DNA. Conversely, extracts prepared at 2 hr or later after vaccinia infection no longer transcribe RNA polymerase II templates but retain the ability to transcribe RNA polymerase III templates as well as vaccinia virus DNA. These profound changes in transcriptional specificity may contribute to the selective expression of viral genes following vaccinia infection.

A vaccinia virus gene that is expressed throughout the reproductive cycle was found to have two sets of RNA start sites approximately 55 nucleotides apart. The site nearest to the coding segment is used early in infection and the one further upstream is used after DNA replication. A series of 5' to 3' deletions were made in the promoter region, and the truncated DNA segments were then ligated to the coding portion of the procaryotic chloramphenicol acetyltransferase gene to measure expression. The effects of these mutations on chloramphenicol acetyltransferase synthesis were determined in a vaccinia virus helper-dependent transient expression system and by forming infectious vaccinia virus recombinants that contain the chimeric genes. Deletions extending up to 31 nucleotides before the late RNA start site had no effect on either early or late expression. Removal of an additional 15 nucleotides produced a dramatic decrease in late expression but had no effect on early expression. The latter was not diminished until the deletion was extended from 31 to 24 nucleotides before the early RNA start site. These results were confirmed by transcriptional analyses. We concluded that this vaccinia virus gene has two promoters and that the regulatory signals for each are located within 31 nucleotides of their sites of transcription.