Related Experiment Video
Updated: Aug 6, 2026
12:01
3' End Sequencing Library Preparation with A-seq2
Published on: October 10, 2017
Asymmetric processing of coding ends and the effect of coding end nucleotide composition on V(D)J recombination
U R Ezekiel1, P Engler, D Stern
1Department of Molecular Genetics and Cell Biology, University of Chicago, Illinois 60637, USA.
Immunity
|April 1, 1995
Summary
Nucleotide composition at coding ends impacts V(D)J recombination. A 5' thymine (T) on recombination signal sequences (RSSs) significantly reduces joint formation, affecting initiation and potentially synapsis.
Area of Science:
- Immunology
- Molecular Biology
- Genetics
Background:
- V(D)J recombination is a crucial process for adaptive immunity, generating diverse antibody and T-cell receptor genes.
- The process involves the joining of V, D, and J gene segments, mediated by the RAG complex.
- Coding and signal joints are the molecular products of this recombination event.
Purpose of the Study:
- To investigate the impact of nucleotide composition at the coding ends of recombination signal sequences (RSSs) on V(D)J recombination.
- To determine how specific nucleotides, particularly thymine (T) and adenine (A), influence coding and signal joint formation.
- To elucidate the mechanistic implications of these nucleotide effects on the V(D)J recombination initiation and processing steps.
Main Methods:
- Analysis of V(D)J recombination efficiency in vitro or in vivo.
- Site-directed mutagenesis to alter nucleotide sequences at the 5' ends of 12-mer and 23-mer RSSs.
- Quantification of coding and signal joint formation using molecular assays (e.g., PCR, sequencing).
Main Results:
- The presence of thymine (T) at the 5' end of either the 12-mer or 23-mer RSS significantly decreases coding and signal joint formation.
- Thymines at the 5' ends of both RSSs abolish V(D)J recombination, indicating an effect on the initiation phase.
- A 5' T coding end can be rescued by the presence of G, C, or A at the other end, suggesting synapsis is required.
- Adenine (A) at the 5' end of the 12-mer RSS affects coding joint formation but not signal joint formation, indicating asymmetric processing.
Conclusions:
- Nucleotide composition of coding ends is a critical determinant of V(D)J recombination efficiency.
- A 5' thymine in RSSs likely interferes with early steps of recombination, possibly RAG complex binding or synaptic complex formation.
- Asymmetric effects of nucleotides on coding versus signal joint formation suggest differential protein complex interactions with the two RSSs during processing.
Related Concept Videos
00:57Viral Recombination
Cells are sometimes infected by more than one virus at once. When two viruses disassemble to expose their genomes for replication in the same cell, similar regions of their genomes can pair together and exchange sequences in a process called recombination. Alternatively, viruses with segmented genomes can swap segments in a process called reassortment.
02:31Homologous Recombination
The basic reaction of homologous recombination (HR) involves two chromatids that contain DNA sequences sharing a significant stretch of identity. One of these sequences uses a strand from another as a template to synthesize DNA in an enzyme-catalyzed reaction. The final product is a novel amalgamation of the two substrates. To ensure an accurate recombination of sequences, HR is restricted to the S and G2 phases of the cell cycle. At these stages, the DNA has been replicated already and the...
02:13Overview of Transposition and Recombination
Transposons make up a significant part of genomes of various organisms. Therefore, it is believed that transposition played a major evolutionary role in speciation by changing genome sizes and modifying gene expression patterns. For example, in bacteria, transposition can lead to conferring antibiotic resistance. Movement of transposable elements within the genetic pool of pathogenic bacteria can aid in transfer of antibiotic-resistant genetic elements. In eukaryotes, transposons can carry out...
02:53Conservative Site-specific Recombination and Phase Variation
Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
The recognition sites for Cre recombinase called LoxP...
The recognition sites for Cre recombinase called LoxP...
01:35Pre-mRNA Processing: Modification of pre-mRNA Ends
In eukaryotic cells, transcripts made by RNA polymerase are modified and processed before exiting the nucleus. Unprocessed RNA is called precursor mRNA or pre-mRNA to distinguish it from mature mRNA.
Once about 20-40 ribonucleotides have been joined together by RNA polymerase, a group of enzymes adds a cap to the 5' end of the growing transcript. In this process, a 5' phosphate is replaced by modified guanosine that has a methyl group attached (7-methyl guanosine). This 5' cap helps the cell...
Once about 20-40 ribonucleotides have been joined together by RNA polymerase, a group of enzymes adds a cap to the 5' end of the growing transcript. In this process, a 5' phosphate is replaced by modified guanosine that has a methyl group attached (7-methyl guanosine). This 5' cap helps the cell...
02:31Homologous Recombination
The basic reaction of homologous recombination (HR) involves two chromatids that contain DNA sequences sharing a significant stretch of identity. One of these sequences uses a strand from another as a template to synthesize DNA in an enzyme-catalyzed reaction. The final product is a novel amalgamation of the two substrates. To ensure an accurate recombination of sequences, HR is restricted to the S and G2 phases of the cell cycle. At these stages, the DNA has been replicated already and the...