(22) and folding of newly translated tubulin (23). In CHO cells the TCP1 complex has been shown to bind newly translated actin and tubulin (25). In Saccharomyces cerevisiae, TCPJ has been shown to be an essential gene, and a cold-sensitive TCP1 mutant exhibited an abnormal appearance of the mitotic spindle (26), supporting a role in the biogenesis of tubulin.To further evaluate the function of the TCP1 complex, we have taken a combination of biochemical and genetic approaches utilizing S. cerevisiae. Here we report the primary structure$ of a second subunit of the complex, TCP1f3, and report on the phenotypes ofgene disruption and temperaturesensitive alleles of both TCPJ (termed here TCPla) and TCP (3. MATERIALS AND METHODSSequence Analysis. The insert of A clone 4950 from a S. cerevisiae chromosome IX library kindly supplied by Maynard Olson and Linda Riles (27) was sequenced (insert of 20,328 bp) after shotgun subcloning of size-selected fragments into M13mpl8 (28). Open reading frames were analyzed with the DIANA program (J. Crooke, T. S. Horsnell, and B.B., unpublished work).Epitope Tagging. Epitopes were added at the C-termini of TCP1a and TCP1(3 immediately upstream from the respective stop codons in coding sequences carried in centromeric (CEN) plasmids YCP50 and YCplac22 (29,30), in the case of a, the influenza hemagglutinin epitope YPYDVPDYA, and for A, the Myc epitope EQKLISEEDL. The hemagglutinin epitope was detected with mouse monoclonal antibody 12CA5 (31) supplied by Berkeley Antibody (Richmond, CA) with permission from Scripps Institute, and the Myc epitope was detected with ascites produced from the hybridoma cell line 9E10 (32), kindly supplied by J. Michael Bishop.Purification of TCP1 Complex from S. cerevisiae. Logarithmically growing cells were harvested and spheroplasts were prepared by zymolyase treatment (33). Spheroplasts were

To further define the transcriptional regulation of the P38 promoter in the minute virus of mice (MVM) genome, we constructed a series of internal deletion and linker scanning mutations. The mutant P38 constructs were assayed for transcriptional activity in vitro by primer extension analysis with nuclear extracts from murine A92L fibroblasts. Mutations which disrupted the GC box and TATA box severely reduced transcription in vitro. DNase I footprinting analysis confirmed that the murine transcription factor Spl bound to the GC box; however, no factors were observed interacting with a putative transcriptional activation regulatory element, termed the TAR element. The linker scanning mutations were analyzed in vivo by using a chloramphenicol acetyltransferase expression assay system, in both the presence and absence of constructs expressing the viral nonstructural protein, NS1. The ability of NS1 to transactivate the P38 promoter (up to 1,000-fold) depended entirely on the presence of intact GC and TATA box sequences. Disruption of the TAR element by either linker insertion mutations or an internal deletion did not inhibit transactivation of the P38 promoter. These results suggest that NS1 transactivates the P38 promoter indirectly by interacting with one or more components of the P38 core-transcription complex.

CDC34 (UBC3) encodes a ubiquitin-conjugating (E2) enzyme required for transition from the G 1 phase to the S phase of the budding yeast cell cycle. CDC34 consists of a 170-residue catalytic N-terminal domain onto which is appended an acidic C-terminal domain. A portable determinant of cell cycle function resides in the C-terminal domain, but determinants for specific function must reside in the N-terminal domain as well. We have explored the utility of ''charge-to-alanine'' scanning mutagenesis to identify novel N-terminal domain mutants of CDC34 that are enzymatically competent with respect to unfacilitated (E3-independent) ubiquitination but that nevertheless are defective with respect to its cell cycle function. Such mutants may reveal determinants of specific in vivo function, such as those required for interaction with substrates or trans-acting regulators of activity and substrate selectivity. Three of 18 ''single-scan'' mutants (in which small clusters of charged residues were mutated to alanine) were compromised with respect to in vivo function. One mutant (cdc34-109, 111, 113A) targeted a 12-residue segment of the Cdc34 protein not found in most other E2s and was unable to complement a cdc34 null mutant at low copy numbers but could complement a null mutant when overexpressed from an induced GAL1 promoter. Combining adjacent pairs of single-scan mutants to produce ''double-scan'' mutants yielded four additional mutants, two of which showed heat and cold sensitivity conditional defects. Most of the mutant proteins expressed in Escherichia coli displayed unfacilitated (E3-independent) ubiquitin-conjugating activity, but two mutants differed from wild-type and other mutant Cdc34 proteins in the extent of multiubiquitination they catalyzed during an autoubiquitination reaction. Our results validate the use of clustered charge-to-alanine scanning mutagenesis for exploring ubiquitin-conjugating enzyme function and have identified additional mutant alleles of CDC34 that will be valuable in further genetic and biochemical studies of Cdc34-dependent ubiquitination.Covalent attachment of the highly conserved protein ubiquitin to other eucaryotic proteins is required for many cellular functions, including stress resistance, selective proteolysis of most normal and abnormal short-lived proteins, cell cycle progression, and DNA repair (14,27,31). Ubiquitination of proteins is catalyzed by an elaborate multienzyme conjugation pathway whose components are conserved in eucaryotes (31). The first step in the ubiquitin ligation pathway is the ATPdependent activation of ubiquitin via the formation of a thiol ester between the ubiquitin C terminus and a cysteine residue in the ubiquitin-activating (E1) enzyme. Ubiquitin is then transferred from the E1 to one of several ubiquitin-conjugating (E2) enzymes, also via a thiol ester formed with a conserved Cys residue. Finally, ubiquitin is transferred from the E2 to a protein substrate in a reaction that often requires a third factor called a ubiquitin protein ligase or E3 (14,...

The P4 promoter of the parvovirus minute virus of mice contains a single degenerate GC box sequence which binds the transcription factor Spl with high affinity. The two protomers of murine Spl were affinity purified, and their interactions with the P4 promoter were examined. Several unusual features were observed. Methylation interference experiments demonstrated that Spl makes contacts with both DNA strands, including the central guanine as well as an adenine residue on the cytidine-rich strand of the GC box. UV photocrosslinking revealed that the 95and the 105-kDa protomers cross-link exclusively to opposite strands of the GC box. These results suggest that the phosphorylation of the 95-kDa Spl protomer results in a change in the way Spl is positioned on the P4 GC box and identifies a high-affinity GC box motif.

Background Single-cell (sc) sequencing performs unbiased profiling of individual cells and enables evaluation of less prevalent cellular populations, often missed using bulk sequencing. However, the scale and the complexity of the sc datasets poses a great challenge in its utility and this problem is further exacerbated when working with larger datasets typically generated by consortium efforts. As the scale of single cell datasets continues to increase exponentially, there is an unmet technological need to develop database platforms that can evaluate key biological hypotheses by querying extensive single-cell datasets. Large single-cell datasets like Human Cell Atlas and COVID-19 cell atlas (collection of annotated sc datasets from various human organs) are excellent resources for profiling target genes involved in human diseases and disorders ranging from oncology, auto-immunity, as well as infectious diseases like COVID-19 caused by SARS-CoV-2 virus. SARS-CoV-2 infections have led to a worldwide pandemic with massive loss of lives, infections exceeding 7 million cases. The virus uses ACE2 and TMPRSS2 as key viral entry associated proteins expressed in human cells for infections. Evaluating the expression profile of key genes in large single-cell datasets can facilitate testing for diagnostics, therapeutics, and vaccine targets, as the world struggles to cope with the on-going spread of COVID-19 infections. Main body In this manuscript we describe REVEAL: SingleCell, which enables storage, retrieval, and rapid query of single-cell datasets inclusive of millions of cells. The array native database described here enables selecting and analyzing cells across multiple studies. Cells can be selected using individual metadata tags, more complex hierarchical ontology filtering, and gene expression threshold ranges, including co-expression of multiple genes. The tags on selected cells can be further evaluated for testing biological hypotheses. One such example includes identifying the most prevalent cell type annotation tag on returned cells. We used REVEAL: SingleCell to evaluate the expression of key SARS-CoV-2 entry associated genes, and queried the current database (2.2 Million cells, 32 projects) to obtain the results in < 60 s. We highlighted cells expressing COVID-19 associated genes are expressed on multiple tissue types, thus in part explains the multi-organ involvement in infected patients observed worldwide during the on-going COVID-19 pandemic. Conclusion In this paper, we introduce the REVEAL: SingleCell database that addresses immediate needs for SARS-CoV-2 research and has the potential to be used more broadly for many precision medicine applications. We used the REVEAL: SingleCell database as a reference to ask questions relevant to drug development and precision medicine regarding cell type and co-expression for genes that encode proteins necessary for SARS-CoV-2 to enter and reproduce in cells.