Recombinant Drosophila melanogaster RPII140-upstream gene protein (140up)

What is the 140up protein in Drosophila melanogaster?

The 140up protein (RPII140-upstream gene protein) is a protein-coding gene product in Drosophila melanogaster that derives its name from its genomic position upstream of the RpII140 gene, which encodes the second-largest subunit (140-kDa) of RNA polymerase II . The protein has a full amino acid sequence of 261 residues beginning with MNFLWKGRRFLIAGILPTFEGAADEIVDKENKTYKAFLASKPPEETGLERLKQMFTIDEF and continuing through the complete sequence as documented in UniProt (P81928) . Its genomic context suggests potential regulatory roles in transcription, though specific molecular functions require further investigation.

Where is the 140up gene located in the Drosophila genome?

The 140up gene is located on chromosome 3R at the polytene band region 88A9-88A9 in Drosophila melanogaster . This chromosomal region contains several other protein-coding genes with which 140up may have functional relationships:

This genomic context is crucial for understanding potential co-regulation patterns and functional relationships between these genes .

What are the known synonyms for the 140up gene?

When conducting literature searches or database queries, researchers should be aware of the various synonyms used for the 140up gene:

• CG9852

• DmRP140-upstream

• DmRP140up

• Dmel\CG9852

• RPII140-upstream gene protein

Using these alternative designations in search strategies ensures comprehensive literature coverage and prevents missing important research findings .

How should recombinant 140up protein be properly stored and handled?

For optimal stability and activity of recombinant Drosophila melanogaster RPII140-upstream gene protein:

• Store stock solutions at -20°C or -80°C for extended storage

• For working aliquots, store at 4°C for up to one week

• Avoid repeated freeze-thaw cycles, which can compromise protein integrity

• Optimal storage buffer typically consists of a Tris-based buffer with 50% glycerol

• For reconstitution, briefly centrifuge vials before opening and use deionized sterile water to achieve a concentration of 0.1-1.0 mg/mL

• Add 5-50% glycerol (final concentration) before aliquoting for long-term storage

The typical shelf life is approximately 6 months for liquid preparations stored at -20°C/-80°C and 12 months for lyophilized preparations under the same conditions .

What expression systems are optimal for producing recombinant 140up protein?

Mammalian cell expression systems have been successfully employed for recombinant production of the 140up protein . This approach offers advantages for a Drosophila protein that may require specific post-translational modifications for proper folding and function.

Methodology for optimal expression:

• Gene synthesis or PCR amplification of the 140up coding sequence

• Cloning into a mammalian expression vector with appropriate promoter and selection markers

• Transfection into mammalian cell lines (e.g., HEK293, CHO)

• Selection of stable cell lines expressing the protein

• Large-scale culture and protein harvest

• Purification using affinity chromatography (facilitated by an appropriate tag)

Expression in bacterial systems may be considered for applications not requiring post-translational modifications, potentially offering higher yields but with possible compromises in protein folding and activity.

How can reverse genetics approaches be applied to study 140up function?

Reverse genetics has proven valuable in studying Drosophila RNA polymerase II subunits and associated proteins. For investigating 140up:

• P-element-mediated transformation: Create transgenic constructs containing the 140up gene with its regulatory regions for rescue experiments or controlled expression

• CRISPR-Cas9 gene editing: Design guide RNAs targeting specific regions of 140up to create precise mutations or gene knockouts

• RNAi approaches: Develop RNA interference constructs to achieve tissue-specific or inducible knockdown of 140up expression

• Creation of deficiency lines: Generate or utilize existing deficiency lines that delete the 88A9-88A9 region to study the effects of 140up loss in conjunction with neighboring genes

• Complementation analysis: Test whether 140up transgenes can rescue lethal phenotypes in mutant lines, as has been done for the neighboring RpII140 gene where a 9.1-kb genomic DNA fragment carried in a P-element construct successfully rescued lethal mutations

What is the relationship between 140up and RpII140?

The genomic arrangement places 140up upstream of the RpII140 gene, which encodes the second-largest subunit of RNA polymerase II in Drosophila melanogaster . This proximity raises several important research considerations:

• Regulatory relationship: The promoter region of RpII140 has been specifically analyzed through sequence comparison between Drosophila melanogaster and Drosophila virilis, suggesting conserved regulatory elements that may also influence 140up expression

• Co-expression patterns: The transcription of DmRP140 has been studied, and researchers should investigate whether 140up and RpII140 show coordinated expression patterns across tissues or developmental stages

• Functional relationship: Given their genomic proximity, 140up may play a role in regulating RpII140 expression or may interact with the RNA polymerase II complex in a manner that affects transcriptional activity

• Evolutionary conservation: The relationship between these genes may be conserved across Drosophila species, suggesting functional importance

What phenotypes are associated with mutations in the 140up gene?

While specific phenotypes for 140up mutations are not directly described in the provided literature, research on the neighboring RpII140 gene provides important context. Mutations in the A5 complementation group, which corresponds to the RpII140 locus, show:

• Specific lethal phases during development

• Interactions with developmental loci such as Ubx (Ultrabithorax)

• Phenotypic similarities to mutations in RpII215, which encodes the largest subunit of RNA polymerase II

Researchers investigating 140up should design experiments to determine:

• Whether null mutations are lethal and at what developmental stage

• Tissue-specific phenotypes using conditional knockouts

• Genetic interactions with neighboring genes including RpII140

• Effects on transcription of specific target genes

• Phenotypic comparisons with RNA polymerase II subunit mutations

How can the interaction between 140up and the transcriptional machinery be studied?

Given its genomic proximity to RpII140, investigating potential interactions between 140up and transcriptional machinery is of significant interest. Methodological approaches include:

• Co-immunoprecipitation (Co-IP): Using antibodies against 140up to pull down associated proteins, followed by mass spectrometry to identify interaction partners within the transcriptional machinery

• Chromatin immunoprecipitation (ChIP): To determine whether 140up associates with chromatin and specific genomic loci, potentially in coordination with RNA polymerase II

• Proximity ligation assays: To visualize in situ interactions between 140up and components of the transcriptional machinery

• Yeast two-hybrid screening: To identify direct protein-protein interactions with components of the transcriptional machinery

• CRISPR-mediated tagging: Endogenous tagging of 140up to allow visualization of its subcellular localization and co-localization with RNA polymerase II components

How does 140up expression change during different developmental stages in Drosophila?

To characterize the developmental expression pattern of 140up:

• RNA-seq analysis: Compare expression levels across embryonic, larval, pupal, and adult stages, and across different tissues

• In situ hybridization: Visualize spatial expression patterns in developing embryos and larval tissues

• Reporter gene constructs: Create transgenic flies with the 140up promoter driving expression of a reporter gene (GFP, LacZ) to monitor expression patterns in vivo

• Quantitative RT-PCR: Measure relative expression levels during specific developmental transitions

• Western blot analysis: Assess protein levels across development using specific antibodies

These approaches would help determine whether 140up expression correlates with key developmental transitions or with expression patterns of RpII140 and other RNA polymerase II components.

Are there homologs of 140up in other model organisms?

Identifying potential homologs in other species requires:

• BLAST analysis: Using the 140up protein sequence to search for similar sequences in other model organisms

• Synteny analysis: Examining whether genes in similar genomic arrangements (upstream of RNA polymerase II subunit genes) exist in other species

• Phylogenetic analysis: Constructing evolutionary trees to determine the relatedness of potential homologs

• Domain conservation: Identifying conserved protein domains or motifs that may indicate functional equivalence despite sequence divergence

• Functional complementation: Testing whether suspected homologs from other species can rescue 140up mutant phenotypes in Drosophila

The genomic organization of RNA polymerase II genes may be conserved across species, and examining this conservation pattern could provide insight into the evolutionary significance of 140up.

How does the promoter region of 140up differ between Drosophila melanogaster and Drosophila virilis?

Previous studies have compared the promoter region of the housekeeping gene DmRP140 between Drosophila melanogaster and Drosophila virilis . Similar analytical approaches can be applied to the 140up promoter:

• Sequence alignment: Identify conserved and divergent regions in the promoter sequences

• Transcription factor binding site prediction: Computational analysis to identify potential regulatory elements

• Reporter gene assays: Test the activity of promoter fragments from both species in transgenic flies

• Chromatin structure analysis: Compare chromatin accessibility and histone modifications at the promoter regions in both species

• Evolutionary rate analysis: Determine whether the promoter has evolved under purifying selection (suggesting functional importance) or shows signs of adaptive evolution

This comparative approach can reveal functionally important regulatory elements and provide insight into the evolutionary conservation of 140up regulation.

What antibodies and detection methods are available for studying 140up expression?

While specific commercial antibodies for 140up are not detailed in the provided search results, researchers can pursue several approaches for protein detection:

• Custom antibody development: Generate antibodies against recombinant 140up protein or synthesized peptide epitopes

• Epitope tagging: Create transgenic flies expressing tagged versions of 140up (e.g., with FLAG, HA, or GFP tags) for detection with commercially available tag antibodies

• Mass spectrometry: For protein identification and quantification in complex samples

• RNA probes: For in situ hybridization to detect 140up mRNA expression patterns

• qRT-PCR primers: Design specific primers for quantitative analysis of 140up transcript levels

When developing detection methods, consider cross-reactivity testing with related proteins and validation in multiple experimental contexts to ensure specificity.

How can bioinformatic tools advance 140up research?

Computational approaches offer powerful methods for generating hypotheses about 140up function:

• Protein structure prediction: Use tools like AlphaFold or I-TASSER to predict 3D structure based on the amino acid sequence

• Protein-protein interaction prediction: Identify potential binding partners through computational docking or co-expression network analysis

• Functional domain identification: Search for conserved domains that might suggest molecular function

• Regulatory network analysis: Integrate expression data to place 140up in gene regulatory networks

• Evolutionary analysis: Compare 140up across Drosophila species to identify conserved regions under selective pressure

These computational approaches can guide experimental design and provide context for interpreting experimental results.

How might CRISPR-Cas9 technology be optimized for studying 140up function?

CRISPR-Cas9 offers precise genome editing capabilities for 140up functional studies:

• Guide RNA design: Create multiple guide RNAs targeting different regions of 140up to generate various alleles (null, hypomorphic, specific domain deletions)

• Homology-directed repair: Introduce precise mutations or reporter genes at the endogenous locus

• Conditional knockout strategies: Implement tissue-specific or temporally controlled CRISPR systems to bypass potential developmental lethality

• Base editing or prime editing: Make specific nucleotide changes without inducing double-strand breaks

• CRISPR activation/repression: Use modified Cas9 systems (dCas9) fused to activators or repressors to modulate 140up expression without altering the sequence

When designing CRISPR experiments, consider potential off-target effects and implement appropriate controls, including rescue experiments with wild-type 140up constructs.

What high-throughput approaches could reveal new insights about 140up function?

Modern high-throughput methodologies offer opportunities for comprehensive functional characterization:

• RNA-seq following 140up manipulation: Identify global transcriptional changes when 140up is overexpressed or depleted

• ChIP-seq: Map genome-wide binding sites if 140up interacts with chromatin

• Proteomics: Identify interaction partners through immunoprecipitation followed by mass spectrometry

• ATAC-seq: Detect changes in chromatin accessibility when 140up function is altered

• Single-cell approaches: Characterize cell-type specific functions of 140up

• Genetic interaction screens: Use CRISPR-based approaches to identify genes that synergize with or suppress 140up phenotypes

These approaches can place 140up in broader biological contexts and generate testable hypotheses about its molecular and cellular functions.