No abstract

Splicing generates mature transcripts from genes in pieces in eukaryotic cells. Overwhelming evidence has accumulated that alternative routes in splicing are possible for most human and mammalian genes, thereby allowing formation of different transcripts from one gene. No function has been assigned to the majority of identified alternative splice forms, and it has been assumed that they compose inert or tolerated waste from aberrant or noisy splicing. Here we demonstrate that five human transcription units (WT1, NOD2, GNAS, RABL2A, RABL2B) have constant splice-isoform ratios in genetically diverse lymphoblastoid cell lines independent of the type of alternative splicing (exon skipping, alternative donor/acceptor, tandem splice sites) and gene expression level. Even splice events that create premature stop codons and potentially trigger nonsense-mediated mRNA decay are found at constant fractions. The analyzed alternative splicing events were qualitatively but not quantitatively conserved in corresponding chimpanzee cell lines. Additionally, subtle splicing at tandem acceptor splice sites (GNAS, RABL2A/B) was highly constrained and strongly depends on the upstream donor sequence content. These results also demonstrate that unusual and unproductive splice variants are produced in a regulated manner. . DNA-encoded genetic information is kept separately in the chromosomes of a eukaryotic nucleus and is decoded via RNA intermediates, which are processed and transmitted to their destinations, for example, to the sites of cytoplasmic protein synthesis. Genes in pieces (composed of exons spaced apart by introns) additionally depend on a splice apparatus (spliceosome) that uses splice signals in a primary transcript to recognize exon-intron boundaries (splice sites) and to accurately cut out introns and join exons. Often, splicing generates different mature transcripts from the same gene, a process called alternative splicing (AS). This is achieved by alternative usage of splice sites in precursor RNA transcripts. In this way, the complexity of transcriptomes and proteomes is increased in eukaryotic organisms. Obviously, AS events need control to ensure formation of proper splice forms and ratios.Sequence motifs matching the splice-site consensus are very common in primary transcripts, but only a minute fraction of them are used by the spliceosome. This specificity is achieved by further sequence and structural information within the premature transcript recognized by different proteins. A complex network of highly combinatorial molecular interactions ensures the tissue-, developmental-, and elicitor-specific formation of spliced transcripts and is regulated at multiple points (Hertel 2008;Smith et al. 2008).Millions of short, single-pass cDNA sequence reads have accumulated in databases as expressed sequence tags supplemented from next-generation transcriptome sequencing data, both of which indicate that AS affects almost any multi-exon gene of any mammalian genome (Mortazavi et al. 2008;Wang et al. 2008).Howev...

Because of the huge diversity, the immunoglobulin repertoire cannot be encoded by static genes, which would explode the genomic capacity comprising about 20,000-25,000 human genes. The immunoglobulin repertoire is provided by the process of somatic germ line recombination, which is the only controlled alteration of the genomic DNA after meiosis. It takes place in mammalian B lymphocyte (B cells) precursors in the bone marrow. The genome germ line sequence of undeveloped B cells is organized in gene segments and compromise V (variable), D (diversity), and J (joining) gene segments constituting the variable domain of the heavy chain and only V and J genes for building up the variable domain of the light chain. The rearrangement of the variable region follows a strict order. The following processes that participate in the generation of antibody diversity were summarized-allelic, combinational, and junctional diversity, pairing of IgH and IgL, and receptor editing-which all together produce the primary antigen repertoire (pre-antigen stimulation). When a B cell encounters a foreign antigen, affinity maturation and class switch are induced. Thereby the antibody repertoire increases. The resulting secondary immunoglobulin repertoire reveals in humans at least 10 11 specificities for different antigens.

In animals and mammalian cells, protein function can be analyzed by nucleotide sequence-based methods such as gene knockout, targeted gene disruption, CRISPR/Cas, TALEN, zinc finger nucleases, or the RNAi technique. Alternatively, protein knockdown approaches are available based on direct interference of the target protein with the inhibitor. Among protein knockdown techniques, the endoplasmic reticulum (ER) intrabodies are potent molecules for protein knockdown in vitro and in vivo. These molecules are increasingly used for protein knockdown in living cells and transgenic mice. ER intrabody knockdown technique is based on the retention of membrane proteins and secretory proteins inside the ER, mediated by recombinant antibody fragments. In contrast to nucleotide sequence-based methods, the intrabody-mediated knockdown acts only on the posttranslational level. In this review, the ER intrabody technology has been compared with the RNAi technique on the molecular level. The generation of intrabodies and RNAi has also been discussed. Specificity and off-target effects (OTE) of these molecules as well as the therapeutic potential of ER intrabodies and RNAi have been compared.