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The neurotransmitter serotonin (5-hydroxytryptamine or 5-HT) mediates rapid excitatory responses through ligand-gated channels (5-HT 3 receptors). Recombinant expression of the only identified receptor subunit (5-HT 3A ) yields functional 5-HT 3 receptors 1 . However, the conductance of these homomeric receptors (sub-picosiemens) is too small to be resolved directly, and contrasts with a robust channel conductance displayed by neuronal 5-HT 3 receptors (9–17 pS) 2 , 3 , 4 , 5 , 6 , 7 . Neuronal 5-HT 3 receptors also display a permeability to calcium ions and a current–voltage relationship that differ from those of homomeric receptors 3 , 4 , 5 , 8 . Here we describe a new class of 5-HT 3 -receptor subunit (5-HT 3B ). Transcripts of this subunit are co-expressed with the 5-HT 3A subunit in the amygdala, caudate and hippocampus. Heteromeric assemblies of 5-HT 3A and 5-HT 3B subunits display a large single-channel conductance (16 pS), low permeability to calcium ions, and a current–voltage relationship which resembles that of characterized neuronal 5-HT 3 channels. The heteromeric receptors also display distinctive pharmacological properties. Surprisingly, the M2 region of the 5-HT 3B subunit lacks any of the structural features that are known to promote the conductance of related receptors. In addition to providing a new target for therapeutic agents, the 5-HT 3B subunit will be a valuable resource for defining the molecular mechanisms of ion-channel function.

The complete 1.66-megabase pair genome sequence of an autotrophic archaeon, Methanococcus jannaschii, and its 58- and 16-kilobase pair extrachromosomal elements have been determined by whole-genome random sequencing. A total of 1738 predicted proteincoding genes were identified; however, only a minority of these (38 percent) could be assigned a putative cellular role with high confidence. Although the majority of genes related to energy production, cell division, and metabolism in M. jannaschii are most similar to those found in Bacteria, most of the genes involved in transcription, translation, and replication in M. jannaschii are more similar to those found in Eukaryotes.

Mast syndrome (SPG21) is an autosomal-recessive complicated form of hereditary spastic paraplegia characterized by dementia, thin corpus callosum, white matter abnormalities, and cerebellar and extrapyramidal signs in addition to spastic paraparesis. A nucleotide insertion resulting in premature truncation of the SPG21 gene product acidic cluster protein 33 (ACP33)/maspardin underlies this disorder, likely causing loss of protein function. However, little is known about the function of maspardin. Here, we report that maspardin localizes prominently to cytoplasm as well as to membranes, possibly at trans -Golgi network/late endosomal compartments. Immunoprecipitation of maspardin with identification of coprecipitating proteins by mass spectrometry revealed the aldehyde dehydrogenase ALDH16A1 as an interacting protein. This interaction was confirmed using overexpressed proteins as well as by fusion protein pull down experiments, and these proteins colocalized in cells. Further studies of the function of ALDH16A1 and the role of the maspardin–ALDH16A1 interaction in neuronal cells may clarify the cellular pathogenesis of Mast syndrome.

Background A recessive mutation “c” in the Mexican axolotl, Ambystoma mexicanum , results in the failure of normal heart development. In homozygous recessive embryos, the hearts do not have organized myofibrils and fail to beat. In our previous studies, we identified a noncoding Myofibril-Inducing RNA (MIR) from axolotls which promotes myofibril formation and rescues heart development. Results We randomly cloned RNAs from fetal human heart. RNA from clone #291 promoted myofibril formation and induced heart development of mutant axolotls in organ culture. This RNA induced expression of cardiac markers in mutant hearts: tropomyosin, troponin and α-syntrophin. This cloned RNA matches in partial sequence alignment to human microRNA-499a and b, although it differs in length. We have concluded that this cloned RNA is unique in its length, but is still related to the microRNA-499 family. We have named this unique RNA, microRNA-499c. Thus, we will refer to this RNA derived from clone #291 as microRNA-499c throughout the rest of the paper. Conclusions This new form, microRNA-499c, plays an important role in cardiac development.

An approach for genome analysis based on sequencing and assembly of unselected pieces of DNA from the whole chromosome has been applied to obtain the complete nucleotide sequence (1,830, 137 base pairs) of the genome from the bacterium Haemophilus influenzae Rd. This approach eliminates the need for initial mapping efforts and is therefore applicable to the vast array of microbial species for which genome maps are unavailable. The H. influenzae Rd genome sequence (Genome Sequence DataBase accession number L42023) represents the only complete genome sequence from a free-living organism.

Mast syndrome (SPG21) is a childhood-onset, autosomal recessive, complicated form of hereditary spastic paraplegia (HSP) characterized by dementia, thin corpus callosum, white matter abnormalities, and cerebellar and extrapyramidal signs in addition to spastic paraparesis. A nucleotide insertion resulting in premature truncation of the SPG21 gene product maspardin underlies this disorder, likely leading to loss of protein function. In this study, we generated SPG21−/− knockout mice by homologous recombination as a possible animal model for SPG21. Though SPG21−/− mice appeared normal at birth, within several months they developed gradually progressive hind limb dysfunction. Cerebral cortical neurons cultured from SPG21−/− mice exhibited significantly more axonal branching than neurons from wild-type animals, while comprehensive neuropathological analysis of SPG21−/− mice did not reveal definitive abnormalities. Since alterations in axon branching have been seen in neurons derived from animal models of other forms of HSP as well as motor neuron diseases, this may represent a common cellular pathogenic theme.

In an effort to identify new genes and analyse their expression patterns, 174,472 partial complementary DNA sequences (expressed sequence tags (ESTs)), totalling more than 52 million nucleotides of human DNA sequence, have been generated from 300 cDNA libraries constructed from 37 distinct organs and tissues. These ESTs have been combined with an additional 118,406 ESTs from the database dbEST, for a total of 83 million nucleotides, and treated as a shotgun sequence assembly project. The assembly process yielded 29,599 distinct tentative human consensus (THC) sequences and 58,384 non-overlapping ESTs. Of these 87,983 distinct sequences, 10,214 further characterize previously known genes based on statistically significant similarity to sequences in the available databases; the remainder identify previously unknown genes. Thirty tissues were sampled by over 1,000 ESTs each; only eight genes were matched by ESTs from all 30 tissues, and 227 genes were represented in 20 or more of the tissues sampled with more than 1,000 ESTs. Approximately 40% of identified human genes appear to be associated with basic energy metabolism, cell structure, homeostasis and cell division, 22% with RNA and protein synthesis and processing, and 12% with cell signalling and communication.

Oncogenic mutations in RAS genes are very common in human cancer, resulting in cells with well-characterized selective advantages, but also less well-understood vulnerabilities. We have carried out a large-scale loss-of-function screen to identify genes that are required by KRAS-transformed colon cancer cells, but not by derivatives lacking this oncogene. Top-scoring genes were then tested in a larger panel of KRAS mutant and wild-type cancer cells. Can- cer cells expressing oncogenic KRAS were found to be highly dependent on the transcription factor GATA2 and the DNA replication initiation regulator CDC6. Extending this analysis using a collection of drugs with known targets, we found that cancer cells with mutant KRAS showed selective addiction to proteasome function, as well as synthetic lethality with topoisomerase inhibition. Combination targeting of these functions caused improved killing of KRAS mutant cells relative to wild-type cells. These observations suggest novel targets and new ways of combining existing therapies for optimal effect in RAS mutant cancers, which are traditionally seen as being highly refractory to therapy.

Photoreceptors in the retina respond to changes in light by modulating the tonic release of glutamate from their axon terminals. Visual information is encoded in the binding of released glutamate to receptors localized to diverse types of bipolar cells that parcel visual information into parallel pathways. Understanding this physiological diversity requires knowledge of the glutamate receptors expressed by bipolar cells. Since glutamate release at the photoreceptor synapse is graded, with the highest concentrations approaching normally toxic levels, bipolar cells might also express glutamate transporters to sharpen their physiological response. I examined the expression of genes encoding glutamate receptors and transporters for the two primary bipolar cell pathways in the retina of the primate Macaque fascicularis: the rod bipolar cell (RBC), which collects rod signals and mediates vision at night, and the diffuse bipolar cell #3 (DB3), which collects from cones and contributes to motion and color vision. RBCs depolarize to light and are known to express the metabotropic glutamate receptor mGluR6, while DB3 cells hyperpolarize to light and are likely to express one or more ionotropic glutamate receptors (GluR). I explored the genetic diversity between RBCs and DB3 cells by creating cDNA libraries from individual cells and probing these libraries using gene-specific PCR, subtractive library hybridization, and random cloning. These libraries were compared to expression in whole retina. I demonstrated for the first time in primate retina genes encoding the AMPA subunits GluR1–4, the kainate subunits GluR5–7 and KA1–2, and the metabotropic subunits mGluR1–8, including a newly described splice variant of mGluR8. I also demonstrated broad expression of the transporters EAAT1–5, including the first evidence of EAAT4 in retina. The RBC expresses the ionotropic subunits GluR1–2 and GluR4, as well as the metabotropic subunits mGluR3 and mGluR6. These cells also express the transporters EAAT2 and EAAT4. In contrast, the DB3 cell expresses only ionotropic subunits GluR1–4 and GluR6, as well the EAAT2 transporter. The results indicate that both pathways for rod and cone vision require not only multiple subunits of glutamate receptors, but also of glutamate transporters to shape their responses to light-induced changes in glutamate concentration.