Recombinant Xenopus laevis Telomere length regulation protein TEL2 homolog (telo2), partial

What is the Xenopus laevis TEL2 homolog and what is its role in telomere regulation?

TEL2 (telomere length regulation protein 2) in Xenopus laevis is a homolog of the yeast Tel2 protein that plays crucial roles in telomere maintenance, genome stability, and DNA damage response. While specific research on Xenopus TEL2 is limited, comparative studies suggest it functions similarly to TEL2 in other organisms. In mammals and yeast, TEL2 forms part of the TTT (TEL2-TTI1-TTI2) complex that assists in the stability and maturation of phosphatidylinositol 3-kinase-related kinases (PIKKs) . These kinases are essential for DNA damage response pathways and cell cycle regulation.

TEL2's specific role in telomere regulation in Xenopus may be connected to factors like XTEF (Xenopus telomere end factor), which specifically recognizes vertebrate telomeric repeats at DNA ends . XTEF has been identified predominantly in Xenopus eggs and ovaries, with an estimated capacity to bind approximately 3×10^7 DNA ends . The relationship between TEL2 and telomere-specific proteins like XTEF represents an important area for further investigation.

How conserved is TEL2 between Xenopus laevis and other model organisms?

TEL2 demonstrates significant evolutionary conservation across species, including yeast, plants, and vertebrates. While the search results don't provide specific sequence comparisons for Xenopus TEL2, we can infer conservation patterns based on other organisms. For instance, the maize TEL2 homolog (ZmTEL2) shares 24.4% protein identity with human TEL2 .

The TEL2 protein typically contains a conserved TEL2 domain along with armadillo repeats, which are likely preserved in the Xenopus homolog. This conservation suggests functional similarities across species. In maize, the TEL2 domain overlaps with an armadillo repeat and is flanked by two additional armadillo repeats , a structural arrangement potentially preserved in Xenopus TEL2.

The functional conservation is demonstrated by cross-species complementation experiments in other systems. For example, in the case of Xenopus Dna2, another DNA maintenance protein, the Xenopus gene was able to complement an S. cerevisiae dna2-1 mutant strain, suggesting functional conservation despite only 32% sequence identity .

What experimental systems are commonly used to study TEL2 function in Xenopus?

Several experimental systems are suitable for investigating TEL2 function in Xenopus:

Egg Extract System: Xenopus egg extracts provide a powerful cell-free system for studying DNA replication and repair processes. This system has been successfully used to study other DNA maintenance proteins like XDna2, where immunodepletion from interphase egg extracts demonstrated its essential role in chromosomal DNA replication . Similar approaches could be applied to study TEL2.

Transgenic Approaches: Multiple transgenic methods have been developed for Xenopus:

Table 1: Comparison of transgenic methods in Xenopus

Cell Culture Systems: Recombinant TEL2 can be expressed in heterologous systems such as insect cells, similar to how XDna2p was expressed and purified for functional studies .

What techniques are available for generating loss-of-function models for TEL2 in Xenopus?

Several approaches can be employed to generate TEL2 loss-of-function models in Xenopus:

Immunodepletion: For rapid functional assessment, TEL2 can be immunodepleted from Xenopus egg extracts. This technique has been successfully applied to study XDna2, where depletion led to almost complete inhibition of chromosomal DNA replication . This approach allows for biochemical assessment of TEL2's immediate functions.

TILLING and Targeted Gene Editing: The diploid Xenopus tropicalis offers enhanced genomics and loss-of-function genetics capabilities. TILLING (Targeting Induced Local Lesions IN Genomes) and targeted gene editing have been established in this species . These techniques could be applied to generate TEL2 mutants.

Morpholino Oligonucleotides: Antisense morpholinos can be used to knockdown TEL2 expression during early development, though results should be interpreted with appropriate controls for off-target effects.

Transgenesis with Dominant Negative Constructs: Using the REMI (Restriction Enzyme Mediated Integration) method, which has an efficiency of over 20% in Xenopus , dominant negative versions of TEL2 could be expressed to interfere with endogenous protein function.

What is known about TEL2 expression patterns during Xenopus development?

While the search results don't provide specific information about TEL2 expression patterns during Xenopus development, we can make inferences based on related proteins. For instance, XTEF (Xenopus telomere end factor) is detected predominantly in extracts of Xenopus eggs and ovaries, with much lower abundance in somatic cell nuclei (approximately 90 per cell) .

To determine TEL2 expression patterns, researchers could employ:

• RT-PCR or qPCR: To quantify TEL2 mRNA levels across developmental stages

• In situ hybridization: To visualize spatial expression patterns

• Western blotting: To assess protein levels in different tissues and developmental timepoints

• Immunohistochemistry: To examine cellular and subcellular localization

The expression pattern would provide insights into developmental contexts where TEL2 may play critical roles in telomere maintenance or DNA damage response.

What are the optimal conditions for expressing and purifying recombinant Xenopus TEL2 protein?

Based on protocols used for similar proteins, the following approach is recommended for recombinant Xenopus TEL2:

Expression System Selection:

• Insect cell expression systems (e.g., baculovirus) have been successfully used for Xenopus DNA maintenance proteins like XDna2p

• Bacterial expression systems may be suitable for specific domains rather than full-length protein

• Mammalian expression systems might preserve post-translational modifications

Cloning Strategy:

• Amplify the full-length TEL2 CDS using PCR primers designed from annotated 5' and 3' UTR regions

• Clone the amplified product into an appropriate entry vector

• Confirm sequence integrity through sequencing

• Subclone into expression vectors with appropriate tags (e.g., His, GST, or FLAG)

Purification Approach:

• Affinity chromatography using tag-specific resins

• Ion exchange chromatography as a secondary purification step

• Size exclusion chromatography for final polishing

• Assess protein quality using SDS-PAGE and Western blotting

For functional characterization, purified TEL2 can be tested for its ability to complement TEL2-deficient systems, similar to how XDna2 complemented S. cerevisiae dna2-1 mutant strains .

How can protein-protein interactions of the TEL2 complex be studied in Xenopus?

Several approaches can be employed to investigate TEL2 protein interactions in Xenopus:

Yeast Two-Hybrid (Y2H):
This approach has been successfully used to study interactions between TEL2, TTI1, and TTI2 in maize . For Xenopus TEL2:

• Clone full-length TEL2 CDS into a bait vector (e.g., pDestDB)

• Clone potential interacting partners into prey vectors (e.g., pDestAD)

• Transform into appropriate yeast strains

• Test for reporter gene activation on selective media

• Confirm interactions with secondary assays

Co-immunoprecipitation (Co-IP):

• Generate antibodies against Xenopus TEL2 or use epitope-tagged versions

• Prepare lysates from Xenopus eggs or tissues

• Immunoprecipitate TEL2 and analyze co-precipitating proteins by Western blot or mass spectrometry

Pull-down Assays:

• Express recombinant TEL2 with affinity tags

• Incubate with Xenopus egg extracts or tissue lysates

• Identify binding partners through mass spectrometry

Proximity Labeling Techniques:
BioID or APEX2 fusions to TEL2 could identify proximal proteins in vivo when expressed in Xenopus embryos through transgenic approaches.

What is the relationship between TEL2 and the TTT complex in Xenopus?

In animals and yeast, TEL2 partners with TTI1 and TTI2 to form the TTT complex, which is involved in stabilizing and maturing PIKKs . While specific data on the Xenopus TTT complex is not provided in the search results, the high conservation of this pathway suggests similar interactions occur in Xenopus.

To investigate the TTT complex in Xenopus:

• Sequence Analysis: Identify Xenopus homologs of TTI1 and TTI2 through bioinformatic approaches

• Expression Analysis: Determine if TEL2, TTI1, and TTI2 are co-expressed in the same tissues and developmental stages

• Interaction Studies: Use co-immunoprecipitation or yeast two-hybrid assays to confirm physical interactions, similar to approaches used for maize TTT components

• Functional Analysis: Assess whether depletion of any TTT component affects the stability of others or their client PIKKs

The TTT complex likely plays critical roles in maintaining genome stability during the rapid cell divisions of early Xenopus development, making it an important focus for research.

How can I assess TEL2's impact on telomere length and stability in Xenopus?

Several approaches can be used to evaluate TEL2's role in telomere maintenance:

Telomere Restriction Fragment (TRF) Analysis:

• Extract high-molecular-weight DNA from control and TEL2-depleted/mutant samples

• Digest with restriction enzymes that do not cut within telomeric sequences

• Perform Southern blotting with telomere-specific probes

• Quantify telomere length distribution

Quantitative FISH (Q-FISH):

• Prepare metaphase chromosome spreads from control and experimental samples

• Hybridize with fluorescent telomere-specific PNA probes

• Quantify fluorescence intensity at chromosome ends

Telomere Dysfunction-Induced Foci (TIF) Analysis:

• Perform immunofluorescence for DNA damage markers (e.g., γ-H2AX)

• Co-stain with telomere probes

• Quantify co-localization to assess telomere dysfunction

Using Xenopus Egg Extracts:
Xenopus egg extracts could be particularly valuable for studying TEL2's role in telomere maintenance. By immunodepleting TEL2 from these extracts and supplementing with DNA templates containing telomeric repeats, researchers could assess changes in telomere processing, similar to approaches used to study XDna2's role in DNA replication .

How does TEL2 function differ between Xenopus laevis and Xenopus tropicalis?

While specific comparative data between X. laevis and X. tropicalis TEL2 is not provided in the search results, several approaches can address this question:

Sequence Comparison:

• Align TEL2 sequences from both species to identify conserved and divergent regions

• Analyze conservation of functional domains and regulatory elements

Expression Pattern Analysis:

• Compare developmental and tissue-specific expression patterns

• Assess relative expression levels in equivalent tissues and developmental stages

Functional Complementation:

• Test whether TEL2 from one species can rescue phenotypes in the other species

• Examine species-specific interaction partners

Experimental Advantages:
X. tropicalis offers specific advantages for TEL2 research including:

• Diploid genome (simplifying genetic analysis)

• Shorter generation time

• Established loss-of-function genetics including TILLING and targeted gene editing

• Smaller genome size facilitating genomic approaches

These comparisons would provide insights into the evolution of TEL2 function and potentially reveal species-specific adaptations.

What experimental approaches can assess TEL2's role in DNA damage response in Xenopus?

Several experimental approaches can evaluate TEL2's involvement in DNA damage response (DDR):

Xenopus Egg Extract System:

• Immunodeplete TEL2 from interphase egg extracts

• Add DNA templates with specific damage (UV-irradiated, oxidized, or containing double-strand breaks)

• Assess DNA repair efficiency and pathway choice

• Monitor activation of checkpoint kinases (ATM, ATR, DNA-PK)

In vivo Approaches:

• Generate TEL2-deficient embryos through morpholinos or CRISPR

• Expose to DNA damaging agents (IR, UV, hydroxyurea)

• Assess survival, developmental defects, and cell cycle checkpoints

• Monitor activation of DDR markers (γ-H2AX, 53BP1, BRCA1)

Biochemical Analysis:

• Examine post-translational modifications of TEL2 following DNA damage

• Identify damage-specific interaction partners

• Assess TEL2's impact on the stability of PIKKs in response to different types of damage

How can I design experiments to investigate potential non-telomeric functions of TEL2 in Xenopus?

Beyond telomere maintenance, TEL2 likely has additional functions that can be investigated through:

Proteomic Approaches:

• Perform immunoprecipitation of TEL2 followed by mass spectrometry to identify interaction partners

• Use proximity labeling (BioID, APEX) to identify proteins in close proximity to TEL2

• Compare interactomes under different conditions (developmental stages, stress conditions)

Transcriptomic Analysis:

• Perform RNA-seq on TEL2-depleted versus control embryos

• Identify pathways dysregulated in the absence of TEL2

• Validate key targets through qRT-PCR and functional assays

Localization Studies:

• Generate fluorescently tagged TEL2 to track subcellular localization

• Examine changes in localization during development or in response to stress

• Identify potential novel sites of action beyond telomeres

Xenopus Double Ovulation Protocol:
For these experiments, researchers can utilize the double ovulation protocol for Xenopus laevis, which produces higher egg yields per animal without compromising egg quality . This approach supports the 3Rs (reduction, refinement, replacement) mission for Xenopus research while providing sufficient material for multiple experimental conditions.