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While voltage-gated potassium channels have critical roles in controlling neuronal excitability, they also have non-ion-conducting functions. Kv8.1, encoded by the KCNV1 gene, is a 'silent' ion channel subunit whose biological role is complex since Kv8.1 subunits do not form functional homotetramers but assemble with Kv2 to modify its ion channel properties. We profiled changes in ion channel expression in amyotrophic lateral sclerosis patient-derived motor neurons carrying a superoxide dismutase 1(A4V) mutation to identify what drives their hyperexcitability. A major change identified was a substantial reduction of KCNV1/Kv8.1 expression, which was also observed in patient-derived neurons with C9orf72 expansion. We then studied the effect of reducing KCNV1/Kv8.1 expression in healthy motor neurons and found it did not change neuronal firing but increased vulnerability to cell death. A transcriptomic analysis revealed dysregulated metabolism and lipid/protein transport pathways in KCNV1/Kv8.1-deficient motor neurons. The increased neuronal vulnerability produced by the loss of KCNV1/Kv8.1 was rescued by knocking down Kv2.2, suggesting a potential Kv2.2-dependent downstream mechanism in cell death. Our study reveals, therefore, unsuspected and distinct roles of Kv8.1 and Kv2.2 in amyotrophic lateral sclerosis-related neurodegeneration., Competing Interests: C.J.W. and K.C.E. are founders of QurAlis Corporation and K.C.E. works at BioMarin Pharmaceutical Inc., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Guarantors of Brain.)

Single cell RNA sequencing methods have been increasingly used to understand cellular heterogeneity. Nevertheless, most of these methods suffer from one or more limitations, such as focusing only on polyadenylated RNA, sequencing of only the 3' end of the transcript, an exuberant fraction of reads mapping to ribosomal RNA, and the unstranded nature of the sequencing data. Here, we developed a novel single cell strand-specific total RNA library preparation method addressing all the aforementioned shortcomings. Our method was validated on a microfluidics system using three different cancer cell lines undergoing a chemical or genetic perturbation and on two other cancer cell lines sorted in microplates. We demonstrate that our total RNA-seq method detects an equal or higher number of genes compared to classic polyA[+] RNA-seq, including novel and non-polyadenylated genes. The obtained RNA expression patterns also recapitulate the expected biological signal. Inherent to total RNA-seq, our method is also able to detect circular RNAs. Taken together, SMARTer single cell total RNA sequencing is very well suited for any single cell sequencing experiment in which transcript level information is needed beyond polyadenylated genes., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Adipose tissue is a rich source of multipotent mesenchymal stem-like cells, located in the perivascular niche. Based on their surface markers, these have been assigned to two main categories: CD31 - /CD45 - /CD34 + /CD146 - cells (adventitial stromal/stem cells [ASCs]) and CD31 - /CD45 - /CD34 - /CD146 + cells (pericytes [PCs]). These populations display heterogeneity of unknown significance. We hypothesized that aldehyde dehydrogenase (ALDH) activity, a functional marker of primitivity, could help to better define ASC and PC subclasses. To this end, the stromal vascular fraction from a human lipoaspirate was simultaneously stained with fluorescent antibodies to CD31, CD45, CD34, and CD146 antigens and the ALDH substrate Aldefluor, then sorted by fluorescence-activated cell sorting. Individual ASCs (n = 67) and PCs (n = 73) selected from the extremities of the ALDH-staining spectrum were transcriptionally profiled by Fluidigm single-cell quantitative polymerase chain reaction for a predefined set (n = 429) of marker genes. To these single-cell data, we applied differential expression and principal component and clustering analysis, as well as an original gene coexpression network reconstruction algorithm. Despite the stochasticity at the single-cell level, covariation of gene expression analysis yielded multiple network connectivity parameters suggesting that these perivascular progenitor cell subclasses possess the following order of maturity: (a) ALDH br ASC (most primitive); (b) ALDH dim ASC; (c) ALDH br PC; (d) ALDH dim PC (least primitive). This order was independently supported by specific combinations of class-specific expressed genes and further confirmed by the analysis of associated signaling pathways. In conclusion, single-cell transcriptional analysis of four populations isolated from fat by surface markers and enzyme activity suggests a developmental hierarchy among perivascular mesenchymal stem cells supported by markers and coexpression networks. Stem Cells 2017;35:1273-1289., (© 2017 AlphaMed Press.)

A novel microRNA (miRNA) quantification method has been developed using stem-loop RT followed by TaqMan PCR analysis. Stem-loop RT primers are better than conventional ones in terms of RT efficiency and specificity. TaqMan miRNA assays are specific for mature miRNAs and discriminate among related miRNAs that differ by as little as one nucleotide. Furthermore, they are not affected by genomic DNA contamination. Precise quantification is achieved routinely with as little as 25 pg of total RNA for most miRNAs. In fact, the high sensitivity, specificity and precision of this method allows for direct analysis of a single cell without nucleic acid purification. Like standard TaqMan gene expression assays, TaqMan miRNA assays exhibit a dynamic range of seven orders of magnitude. Quantification of five miRNAs in seven mouse tissues showed variation from less than 10 to more than 30,000 copies per cell. This method enables fast, accurate and sensitive miRNA expression profiling and can identify and monitor potential biomarkers specific to tissues or diseases. Stem-loop RT-PCR can be used for the quantification of other small RNA molecules such as short interfering RNAs (siRNAs). Furthermore, the concept of stem-loop RT primer design could be applied in small RNA cloning and multiplex assays for better specificity and efficiency.

We have prepared and evaluated a series of structural analogues of TaqMan PCR probes in an effort to identify second-generation probes with improved physical properties and performance. Modifications have included non-nucleosidic dye linkers, 2'-O-Me RNA substitutions, and pyrimidine C5-propyne substitutions.

A simple primer extension method for detecting nucleotide differences is based on the substitution of mobility-shifting analogs for natural nucleotides (1). This technique can detect any single-base difference that might occur including previously unknown mutations or polymorphisms. Two technical limitations of the original procedure have now been addressed. First, switching to Thermococcus litoralis DNA polymerase has eliminated variability believed to be due to the addition of an extra, non-templated base to the 3' end of DNA by Taq DNA polymerase. Second, with the analogs used in the original study, the mobility shift induced by a single base change can usually be resolved only in DNA segments 200 nt or smaller. This size limitation has been overcome by synthesizing biotinylated nucleotides with extraordinarily long linker arms (36 atom backbone). Using these new analogs and conventional sequencing gels (0.4 mm thick), mutations in the human beta-hexosaminidase alpha and CYP2D6 genes have been detected in DNA segments up to 300 nt in length. By using very thin (0.15 mm) gels, single-base polymorphisms in the human APOE gene have been detected in 500-nt segments.

Expression of the X-linked repeated Stellate (Ste) genes, which code for a protein with 38% similarity to the beta-subunit of casein kinase II, is suppressed by the Su(Ste) locus on the Y chromosome. The structure and evolution of the Y-linked repeats in the region of the Su(Ste) locus were studied. The 2800 bp repeats consist of three main elements: the region of homology to the Ste genes, an adjacent AT-rich, Y-specific segment, and mobile element 1360 inserted in the Ste sequence. Amplification of repeats was followed by point mutations, deletions, and insertions of mobile elements. DNA sequencing shows that these repeats may be considered as Ste pseudogenes or as damaged variants of a putative gene(s) encoding a protein quite different from the Ste protein as a result of an alternative splicing pattern. A comparison of 5 variants of the Y-Su(Ste) repeats shows a number of recombination events between amplified and diverged sequences that could be due to either multiple unequal mitotic sister-chromatid exchanges or to gene conversion. It is a first demonstration on a molecular level of these processes occurring in heterochromatic non-rDNA tandemly organized sequences in an eukaryotic genome.

Molecular genetic maps are commonly constructed by analyzing the segregation of restriction fragment length polymorphisms (RFLPs) among the progeny of a sexual cross. Here we describe a new DNA polymorphism assay based on the amplification of random DNA segments with single primers of arbitrary nucleotide sequence. These polymorphisms, simply detected as DNA segments which amplify from one parent but not the other, are inherited in a Mendelian fashion and can be used to construct genetic maps in a variety of species. We suggest that these polymorphisms be called RAPD markers, after Random Amplified Polymorphic DNA.

The X-linked Stellate locus contains two major size classes of a tandemly repeated gene. An example of each class has been sequenced. The steady-state level of Stellate RNA is much higher in XO testis than in XY testis. Sequencing of six cDNA clones derived from XO testis RNA shows that there are two major introns in the Stellate genes. Primer extension and RNase protection analyses show that these introns are spliced much more efficiently in XO than in XY testis. These results also indicate the major transcriptional start site for Stellate RNA. P element transformation results with a marked Stellate gene demonstrate that at least one of the genes sequenced contains a functional promoter, which generates low levels of RNA in XY testis and high levels of RNA in XO testis. This promoter does not contain a TATA element in the -30 region relative to the transcriptional start. Previous results had implicated a specific region of the Y chromosome, designated here as the Su(Ste) locus, in the control of the Stellate genes on the X. Analysis using segmental Y deficiencies shows that the Su(Ste) region suppresses both the high levels and efficient splicing of Stellate RNA.

Males carrying a large deficiency in the long arm of the Y chromosome known to delete the fertility gene kl-2 are sterile and exhibit a complex phenotype: (1) First metaphase chromosomes are irregular in outline and appear sticky; (2) spermatids contain micronuclei; (3) the nebenkerns of the spermatids are nonuniform in size; (4) a high molecular weight protein ordinarily present in sperm is absent; and (5) crystals appear in the nucleus and cytoplasm of spermatocytes and spermatids. In such males that carry Ste+ on their X chromosome the crystals appear long and needle shaped; in Ste males the needles are much shorter and assemble into star-shaped aggregates. The large deficiency may be subdivided into two shorter component deficiencies. The more distal is male sterile and lacks the high molecular weight polypeptide; the more proximal is responsible for the remainder of the phenotype. Ste males carrying the more proximal component deficiency are sterile, but Ste+ males are fertile. Genetic studies of chromosome segregation in such males reveal that (1) both the sex chromosomes and the large autosomes undergo nondisjunction, (2) the fourth chromosomes disjoin regularly, (3) sex chromosome nondisjunction is more frequent in cells in which the second or third chromosomes nondisjoin than in cells in which autosomal disjunction is regular, (4) in doubly exceptional cells, the sex chromosomes tend to segregate to the opposite pole from the autosomes and (5) there is meiotic drive; i.e., reciprocal meiotic products are not recovered with equal frequencies, complements with fewer chromosomes being recovered more frequently than those with more chromosomes. The proximal component deficiency can itself be further subdivided into two smaller component deficiencies, both of which have nearly normal spermatogenic phenotypes as observed in the light microscope. Meiosis in Ste+ males carrying either of these small Y deficiencies is normal; Ste males, however, exhibit low levels of sex chromosome nondisjunction with either deficient Y. The meiotic phenotype is apparently sensitive to the amount of Y chromosome missing and to the Ste constitution of the X chromosome.

The genome of the virus associated with the acquired immune deficiency syndrome (AIDS), human T-lymphotropic virus type III (HTLV-III), includes two open reading frames, not found in other retroviruses. One of these, designated 3' open reading frame (3'orf) is 648 base pairs (bp) in length, and overlaps with the 3' long terminal repeat (LTR) sequences. Sequences of additional HTLV-III clones were determined in order to estimate the level and location of variation within 3'orf, to gain some insight into the function of its protein product. Newly determined sequences are reported for 3'orf of two unintegrated clones of HTLV-III and three cDNA clones made from virion RNA derived from the same cell line infected with pooled blood samples of different patients with AIDS or AIDS-related complex symptoms (ARC). In addition, sequences for 3'orf were derived from an unintegrated viral clone derived from a different cell line infected with a distinct isolate from a single patient. These sequences are compared to those previously reported for six other viral clones. Sequences of 3'orf differ among clones by 1.1-10.4% bp and 2.4-17.0% of predicted amino acids. This represents significantly greater sequence variation than is found in the entire genome on average. Moreover, a functional proviral clone has a termination codon at amino acid residue 124 of this open reading frame. This raises questions concerning the structure, and regulation of expression of the protein encoded by 3'orf.

The D. melanogaster DNA segment in the recombinant phage lambda Dm2L1 contains at least eight copies of a tandemly repeated 1250-base pair (bp) sequence (henceforth called the 2L1 sequence). Testes from XO D. melanogaster males contain an abundant 800-base RNA species that is homologous to a 520-bp region of the 2L1 sequence. Blotting experiments show that the 2L1 sequence is repeated in the D. melanogaster genome and is present on both the X and Y chromosomes. With the use of X-Y translocations, the 2L1 sequence has been mapped to a region between kl-1 and kl-2 on the long arm of the Y chromosome. In Oregon-R wild type there are an estimated 200 copies of the 2L1 sequence on the X chromosome and probably at least 80 copies of the Y chromosome. In some other strains the repetition frequency on the Y chromosome is about the same, but the copy number on the X chromosome is much reduced. On the basis of the five strains investigated, there is a correlation between copy number of the 2L1 sequence on the X chromosome and the presence of a particular allele of the Stellate locus (Ste; 1-45.7). It seems that low copy number corresponds to Ste+ and high copy number corresponds to Ste. The Ste locus determines whether single or star-shaped crystals are observed in the spermatocytes of XO males. Studies using D. simulans and D. mauritiana DNA show that the 2L1 sequence is homologous to restriction fragments in male DNA but not female DNA, indicating that this sequence is present only on the Y chromosome in these two species. In DNA derived from D. erecta, D. teissieri and D. yakuba, there is very little, if any, hybridization with the 2L1 sequence probe.

A simple primer extension assay has been developed to distinguish homologous DNA segments differing by as little as a single nucleotide. DNA strands are synthesized with one of the four natural nucleotides replaced with an analog that affects electrophoretic mobility. DNAs that are the same length but differ in the number of analog molecules per strand exhibit different mobilities on a sequencing gel. In combination with the polymerase chain reaction (PCR; 1, 2), this method has been used to distinguish mutant and normal alleles of the human insulin receptor gene that differ by a single-base substitution. The method appears to be generally applicable to the detection of any nucleotide polymorphism in any segment of DNA.

We have determined the nucleotide sequence of the yeast ILV2 gene which codes for the amino acid biosynthetic enzyme acetolactate synthase (ALS). ALS has recently been shown to be the target in bacteria, yeast and plants, of the potent new herbicide sulfometuron methyl. The coding sequence for the ILV2 polypeptide contains 2061 base pairs. Comparison of deduced amino acid sequences indicates considerable conservation between the yeast protein and the large subunits of the E. coli ALS II and ALS III isozymes. A major distinction between the three proteins is the presence of an additional 90 amino acids at the amino terminal of the yeast protein. The amino acid sequence in this region shows similarities to yeast mitochondrial transit sequences and may function as such, since yeast ALS is localized in the mitochondria. Consensus sequences for initiation and termination of transcription that are consistent with the ends of the ILV2 mRNA, as well as general amino acid control regulatory sequences have been identified.