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Query: UNIPROT:P06889 (Mol)
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Introns are generally believed to evolve too rapidly and too erratically to be of much use in phylogenetic reconstructions. Few phylogenetically informative intron sequences are available, however, to ascertain the validity of this supposition. In the present study the supposition was tested on the example of the mammalian class II major histocompatibility complex (Mhc) genes of the DRB family. Since the Mhc genes evolve under balancing selection and are believed to recombine or rearrange frequently, the evolution of their introns could be expected to be particularly rapid and subject to scrambling. Sequences of intron 4 and 5 DRB genes were obtained from polymerase chain reaction-amplified fragments of genomic DNA from representatives of six eutherian orders-Primates, Scandentia, Chiroptera, Dermoptera, Lagomorpha, and Insectivora. Although short stretches of the introns have indeed proved to be unalignable, the bulk of the intron sequences from all six orders, spanning >85 million years (my) of evolution, could be aligned and used in a study of the tempo and mode of intron evolution. The analysis has revealed the Mhc introns to evolve at a rate similar to that of other genes and of synonymous sites of non-Mhc genes. No evidence of homogenization or large-scale scrambling of the intron sequences could be found. The Mhc introns apparently evolve largely by point mutations and insertions/deletions. The phylogenetic signals contained in the intron sequences could be used to identify Scandentia as the sister group of Primates, to support the existence of the Archonta superorder, and to confirm the monophyly of the Chiroptera.
J Mol Evol 1999 Jun
PMID:Evolution of Mhc-DRB introns: implications for the origin of primates. 1022 70

Background: Many genetic loci exhibit substantial heterogeneity: the human leukocyte antigen (HLA) DRB loci include 139 alleles and the cystic fibrosis transmembrane regulator gene more than 500 known mutations. Identification of alleles at these loci is cumbersome with typical molecular diagnostic methods such as hybridization assays or restriction enzyme analysis. Direct DNA sequencing of polymerase chain reaction (PCR) products is a general approach to complex loci that allows detection of any allele within the nucleotide sequence analyzed. However, direct DNA sequence-based unambiguous identification of heterozygous nucleotide positions using PCR templates is a challenging problem. Methods and Results: The ability of direct DNA sequencing methods to accurately identify HLA DRB alleles was assessed. The authors evaluated the performance of modified T7 and Taq DNA polymerases in isothermal and thermal cycle sequencing of PCR products derived from HLA DRB genes in 235 individuals who were potential donors or recipients of bone marrow transplants. The uniformity of peak intensity and ability to identify heterozygous nucleotide positions was similar when either AmpliTaq FS- or Sequenase DNA polymerase-derived electropherograms were prepared. The modified Taq DNA polymerase allowed the use of unpurified, double-stranded PCR templates. Furthermore, this enzyme could be used in less laborious, less costly cycle sequencing assays coupled with automated fluorescent detection methodology. Direct sequencing performed with either enzyme allowed unambiguous identification of DRB1 alleles, resolution of difficult heterozygous combinations, and recognition of new alleles. Conclusions: The direct DNA sequencing methods employed here for HLA allele identification are relatively efficient and semiautomated, and may be reasonably considered as a general approach to other complex molecular diagnostic problems, especially when coupled to simplified sequencing chemistries allowing cycle sequencing.
Mol Diagn 1996 Jun
PMID:Strategies for Unambiguous Detection of Allelic Heterozygosity via Direct DNA Sequencing of PCR Products: Application to the HLA DRB1 Locus. 1033 Feb 4

Exact human lymphocyte antigen (HLA) allele matching and sequence variation are important for matching organ donors, immune response studies, and disease association investigations. The number of HLAs has reached several hundred within each of the different classes. This level of heterogeneity makes routine DNA typing to the allele level problematic using fixed probe and primer technologies. Routine large-batch screening programs where only intermediate-level typing is required can be performed in automated fashion by several DNA technologies. Screening large numbers of samples with probe-based technologies, even oligo array chips, is cost effective. Allele-specific typing is most easily performed using direct sequencing. New sequencing technologies based on cycle sequencing and high-speed capillary gels have made routine sequencing for clinical typing a reality. The complexity of the class I locus requires a detailed analysis of all the polymorphisms within exons 2 and 3. Sequencing strategies are thus designed to use informative variable regions within the flanking introns and the flanking region as well as the untranslated regions. Simliar strategies are being adapted to the complex class II DRB alleles, which now number about 200 different alleles. Greater understanding of HLA diversity and distribution throughout humans and their relatives facilitates organ matching and the history and origins of human populations. Knowledge of parasites and their role in the selection of alleles will ultimately lead to better prediction and manipulation of the immune system response to these organisms. HLA typing is used to determine relative risk to a variety of autoimmune diseases. Future uses of molecular HLA typing may include the prevention and cessation of these self-destructing diseases.
Mol Diagn 1997 Dec
PMID:Human Lymphocyte Antigen Typing: Direct DNA Typing-The Only Choice? 1046 21

We report that the chromatin-specific transcription elongation factor FACT functions in conjunction with the RNA polymerase II CTD kinase P-TEFb to alleviate transcription inhibition by DSIF (DRB sensitivity-inducing factor) and NELF (negative elongation factor). We find that the kinase activity of TFIIH is dispensable for this activity, demonstrating that TFIIH-mediated CTD phosphorylation is not involved in the regulation of FACT and DSIF/NELF activities. Thus, we propose a novel transcriptional regulatory network in which DSIF/NELF inhibition of transcription is prevented by P-TEFb in cooperation with FACT. This study uncovers a novel role for FACT in the regulation of transcription on naked DNA that is independent of its activities on chromatin templates. In addition, this study reveals functional differences between P-TEFb and TFIIH in the regulation of transcription.
Mol Cell 2000 Jun
PMID:FACT relieves DSIF/NELF-mediated inhibition of transcriptional elongation and reveals functional differences between P-TEFb and TFIIH. 1091 1

A New World monkey, the common marmoset (Callithrix jacchus), will be used as a preclinical animal model to study the feasibility of cell and gene therapy targeting immunological and hematological disorders. For elucidating the immunogenetic background of common marmoset to further studies, in the present study, polymorphisms of MHC-DRB genes in this species were examined. Twenty-one Caja-DRB exon 2 alleles, including seven new ones, were detected by means of subcloning and the polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) methods followed by nucleotide sequencing. Based on the alignment of these allele sequences, we designed two pairs of specific primers and established a PCR-SSCP method for DNA-based histocompatibility typing of the common marmoset. According to the family segregation data and phylogenetic analyses, we presumed that Caja-DRB alleles could be classified into five different loci. Southern blotting analysis also supported the existence of multiple DRB loci. The patterns of nucleotide substitutions suggests that positive selection operates in the antigen-recognition sites of Caja-DRB genes.
J Mol Evol 2000 Sep
PMID:MHC (major histocompatibility complex)-DRB genes and polymorphisms in common marmoset. 1102 66

Major histocompatibility complex (MHC) class II locus DRB was investigated by single-strand conformation polymorphism analysis (SSCP) and sequence analysis in the endemic South African antelope, Damaliscus pygargus, of which there are two subspecies. Greater polymorphism was found in the blesbok (D. p. phillipsi) subspecies (n = 44; 22 alleles) than in the bontebok (D. p. pygargus) subspecies (n = 45; 6 alleles). Erosion of allelic diversity in bontebok was most likely the result of two severe bottleneck events caused by hunting pressure and parasitic infection. A majority of the polymorphism observed was found within the peptide binding region (PBR) where dN/dS ratios were higher than for the non-PBR region. This, and the apparent trans-species relationship among alleles in a bovid phylogeny, suggest the evolution of diversity by heterosis or frequency-dependent selection.
Mol Ecol 2001 Jul
PMID:Characterization of major histocompatibility complex DRB diversity in the endemic South African antelope Damaliscus pygargus: a comparison in two subspecies with different demographic histories. 1147 36

Expression of the retinoblastoma tumor suppressor protein (Rb) is required for gamma interferon (IFN-gamma)-inducible major histocompatibility complex class II gene expression and transcriptionally productive HLA-DRA promoter occupancy in several human tumor cell lines. Treatment of these Rb-defective tumor cell lines with histone deacetylase (HDAC) inhibitors rescued IFN-gamma-inducible HLA-DRA and -DRB mRNA and cell surface protein expression, demonstrating repression of these genes by endogenous cellular HDAC activity. Additionally, Rb-defective, transcriptionally incompetent tumor cells retained the HLA-DRA promoter DNase I-hypersensitive site. Thus, HDAC-mediated repression of the HLA-DRA promoter occurs following the establishment of an apparent nucleosome-free promoter region and before transcriptionally productive occupancy of the promoter by the required transactivators. Repression of HLA-DRA promoter activation by HDAC activity likely involves a YY1 binding element located in the first exon of the HLA-DRA gene. Chromatin immunoprecipitation experiments localized YY1 to the HLA-DRA gene in Rb-defective tumor cells. Additionally, mutation of the YY1 binding site prevented repression of the promoter by HDAC1 and partially prevented activation of the promoter by trichostatin A. Mutation of the octamer element also significantly reduced the ability of HDAC1 to confer repression of inducible HLA-DRA promoter activation. Treatment of Rb-defective tumor cells with HDAC inhibitors greatly reduced the DNA binding activity of Oct-1, a repressor of inducible HLA-DRA promoter activation. These findings represent the first evidence that HDAC activity can repress IFN-gamma-inducible HLA class II gene expression and also demonstrate that HDAC activity can contribute to promoter repression following the establishment of a DNase I-hypersensitive chromatin conformation.
Mol Cell Biol 2001 Oct
PMID:Histone deacetylase activity represses gamma interferon-inducible HLA-DR gene expression following the establishment of a DNase I-hypersensitive chromatin conformation. 1153 38

To stimulate transcriptional elongation of HIV-1 genes, the transactivator Tat recruits the positive transcription elongation factor b (P-TEFb) to the initiating RNA polymerase II (RNAPII). We found that the activation of transcription by RelA also depends on P-TEFb. Similar to Tat, RelA activated transcription when tethered to RNA. Moreover, TNF-alpha triggered the recruitment of P-TEFb to the NF-kappaB-regulated IL-8 gene. While the formation of the transcription preinitiation complex (PIC) remained unaffected, DRB, an inhibitor of P-TEFb, prevented RNAPII from elongating on the IL-8 gene. Remarkably, DRB inhibition sensitized cells to TNF-alpha-induced apoptosis. Thus, NF-kappaB requires P-TEFb to stimulate the elongation of transcription and P-TEFb plays an unexpected role in regulating apoptosis.
Mol Cell 2001 Aug
PMID:NF-kappaB binds P-TEFb to stimulate transcriptional elongation by RNA polymerase II. 1154 35

We identify and characterize several phosphorylated forms of the hSpt5 subunit of the DRB sensitivity-inducing factor (DSIF). A 175-kDa phosphorylated form of hSpt5 is bound to nuclei of interphase HeLa cells. This form is rapidly dephosphorylated when cultured cells are exposed to various drugs belonging to distinct chemical families. All these compounds are known to inhibit the protein kinase Cdk9, which phosphorylates in vitro hSpt5 and Rpb1, the largest subunit of RNA polymerase II. The efficiency to promote the dephosphorylation of both proteins matches their capacity to inhibit purified Cdk9 kinase, suggesting that Cdk9 is the major kinase phosphorylating hSpt5 and Rpb1 in vivo. We show that Cdk9 phosphorylates both the CTR1 and the CTR2 domains of recombinant hSpt5. These domains contain numerous serine-proline and threonine-proline residues similar to those found in the carboxyl-terminal domain (CTD) of Rpb1. The structural homology between hSpt5 CTRs and the Rpb1 CTD is further highlighted by the presence on both proteins of a phosphoepitope recognized by the monoclonal antibody CC-3. Of particular interest, the peptidyl-prolyl isomerase Pin1 interacts with Cdk9-phosphorylated hSpt5. Cdk9 dependent phosphorylation of Rpb1 and hSpt5 followed by Pin1 interaction might thus contribute to the regulation of transcription, pre-mRNA maturation, and the dynamics of these proteins in interphase and mitosis.
J Mol Biol 2001 Sep 28
PMID:The peptidyl-prolyl isomerase Pin1 interacts with hSpt5 phosphorylated by Cdk9. 1157 23

Negative elongation factor (NELF) is a human transcription factor complex that cooperates with DRB sensitivity-inducing factor (DSIF)/hSpt4-hSpt5 to repress elongation by RNA polymerase II (RNAPII). NELF activity is associated with five polypeptides, including NELF-A, a candidate gene product for Wolf-Hirschhorn syndrome, and NELF-E, a putative RNA-binding protein with arginine-aspartic acid (RD) dipeptide repeats. Here we report several important findings regarding the DSIF/NELF-dependent elongation control. First, we have established an effective method for purifying the active NELF complex using an epitope-tagging technique. Second, the five polypeptides each are important and together are sufficient for its function in vitro. Third, NELF does not bind to either DSIF or RNAPII alone but does bind to the preformed DSIF/RNAPII complex. Fourth, NELF-E has a functional RNA-binding domain, whose mutations impair transcription repression without affecting known protein-protein interactions. Taken together, we propose that NELF causes RNAPII pausing through binding to the DSIF/RNAPII complex and to nascent transcripts. These results also have implications for how DSIF and NELF are regulated in a gene-specific manner in vivo.
Mol Cell Biol 2002 May
PMID:Evidence that negative elongation factor represses transcription elongation through binding to a DRB sensitivity-inducing factor/RNA polymerase II complex and RNA. 1194 Jun 50


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