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Biomedical subjects

Z Yao

Publications and source records attributed to Z Yao.

At least 163 records · Page 9Linked to original sources
PubMed1996

Apolipoprotein B sequence requirements for hepatic very low density lipoprotein assembly. Evidence that hydrophobic sequences within apolipoprotein B48 mediate lipid recruitment.

We studied the structural requirements of apolipoprotein (apo) B for assembly of very low density lipoproteins (VLDL) using rat hepatoma McA-RH7777 cells expressing human apoB (h-apoB). Recombinant h-apoB48, like endogenous rat apoB48 (r-apoB48), was secreted as VLDL in addition to high density lipoproteins (HDL) by transfected cells, indicating that the N-terminal 48% of apoB contains sequences sufficient for VLDL assembly. Truncation of the C terminus of h-apo-B48 to -B42 or -B37 had little effect on the ability of apoB to assemble VLDL, whereas truncation to -B34 or -B29 markedly diminished or abolished VLDL formation. None of the truncations affected the integration of apoB into HDL. To determine whether the ability to assemble VLDL is governed by apoB length or by sequences beyond apoB29, we created chimeric proteins that contained human apoA-I and a segment derived from between the C-terminal 29 and 34%, 34 and 37%, or 37 and 42% of apoB100. The resulting chimeras, namely AI/B29-34, AI/B34-37, and AI/B37-42, were secreted by the transfected cells as lipoproteins with buoyant density (d < 1.006 g/ml), electrophoretic mobility (pre-beta), and size characteristics of human plasma VLDL. The chimeras could assemble discrete VLDL particles devoid of endogenous r-apoB100, and could actively recruit triglycerides and phospholipids into the lipoproteins. However, these chimeras were secreted inefficiently. Pulse-chase analysis showed that less than 5% of the newly synthesized AI/B proteins were secreted, and more than 70% was degraded intracellularly. Degradation of the chimeras could be blocked by the cysteine protease inhibitor N-acetyl-leucyl-leucyl-norleucinal, but the treatment did not enhance their secretion. Protease protection analysis of microsomes isolated from transfected cells indicated that >65% of AI/B chimeras (compared with <25% of r-apoB100) were inaccessible to exogenous trypsin. These data suggest that the recruitment of large quantities of triglycerides during VLDL formation is not governed simply by apoB length, but is mediated by short hydrophobic sequences ranging from 152 to 237 amino acids (3-5%) of apoB. The existence of multiple such hydrophobic sequences within apoB48 may facilitate efficient assembly of hepatic VLDL particles.

Animals↗
PubMed1996

The microsomal triglyceride transfer protein facilitates assembly and secretion of apolipoprotein B-containing lipoproteins and decreases cotranslational degradation of apolipoprotein B in transfected COS-7 cells.

We studied the role of microsomal triglyceride transfer protein (MTP) in the synthesis, secretion, and cotranslational degradation of apolipoprotein (apo) B using nonhepatic COS-7 cells that expressed C-terminally truncated forms of apoB (from apoB15 to apoB94) with or without the large subunit of human MTP. With the exception of apoB15 and apoB18, secretion of all of the apoB forms was stimulated by expression of MTP, even though a small amount of short apoB forms (</=apoB48) could be secreted by cells transfected with apoB alone. The majority of the apoB protein, including apoB72 and apoB94, was secreted as high density lipoprotein (1.08-1.17 g/ml). Pulse-chase experiments revealed that the secretion efficiency of apoB94 and apoB72 was low (ranging from 2 to 12%). The failure to secrete buoyant lipoproteins and the low secretion efficiency were associated with insufficient lipid synthesis by the cells. The incorporation of [3H]oleate into cellular triglyceride and phosphatidylcholine by COS cells over a 2-h period was 28 and 38%, respectively, of that by rat hepatoma (McA-RH7777) cells. In addition to the desired full-length apoB, cells transfected with large constructs (>/=apoB60) also produced smaller species with a size of approximately220 kDa (designated B48-like protein). Coexpression with MTP decreased formation of the B48-like proteins by 40-60%. The reduction in B48-like protein formation was specific to MTP expression; coexpression with other proteins (e.g. apoA-I or apoB15) did not alter B48-like protein production. Kinetic analysis suggested that B48-like proteins were produced concurrently (cotranslational) with the full-length apoB94 and apoB72 and were not products of post-translational degradation. Although some of the B48-like proteins might be derived from truncated species (approximately 7 kb in size) of apoB mRNA that were found in cells transfected with large apoB constructs, MTP coexpression did not affect the relative levels of the aberrant 7-kb RNA with respect to the full-length mRNA. However, coexpression of MTP decreased the accessibility of apoB to exogenous trypsin by 2-fold for apoB72 and by 10-fold for apoB94 in isolated microsomes. Thus, the reduced B48-like protein formation by MTP may be a consequence of attenuated cotranslational degradation during apoB translocation across the ER membrane. Formation of B48-like proteins was insensitive to N-acetyl-leucyl-leucyl-norleucinal, a cysteine protease inhibitor known to block post-translational degradation of apoB. These results indicate that MTP facilitates the assembly and secretion of lipoproteins containing apoB and also attenuates the formation of B48-like proteins, probably by assisting apoB translocation across the ER membrane.

Animals↗
PubMed1996

Herpesvirus saimiri open reading frame 14, a protein encoded by T lymphotropic herpesvirus, binds to MHC class II molecules and stimulates T cell proliferation.

Herpesvirus saimiri (HVS) is an oncogenic, lymphotropic, gamma-herpesvirus that transforms human and simian T cells in vitro and causes lymphomas and leukemias in various species of New World primates. Nucleotide sequence analysis of the HVS genome revealed an open reading frame with 22% amino acid identity to the mouse mammary tumor virus 7 superantigen. In this study, we demonstrate that this open reading frame, HVS14, encodes a heavily glycosylated protein that is secreted. Both the HVS14 present in the supernatant of transfected cells and a chimeric HVS14.Fc fusion protein were found to bind to heterodimeric MHC class II HLA-DR molecules. The supernatant from HVS14-transfected cells induced the proliferation of human PBMC, which could be specifically inhibited by HVS14-specific mAbs. Purified peripheral blood T cells were induced to proliferate in the presence of accessory cells and HVS14-containing supernatant. Whereas the HVS14 protein stimulated T cell proliferation, the HVS14.Fc fusion protein blocked proliferative responses to soluble Ags in vitro. Collectively, these data indicate that HVS14 can function as an immunomodulator that may contribute to the immunopathology of HVS infection.

Cell-Free System↗
PubMed1996

The amino terminus of apolipoprotein B is necessary but not sufficient for microsomal triglyceride transfer protein responsiveness.

Human apolipoprotein (apo) B mediates the formation of neutral lipid-containing lipoproteins in the liver and intestine. The association of apoB with lipid is thought to be promoted by the microsomal triglyceride transfer protein complex. We have reconstituted lipoprotein assembly in an insect cell line that normally does not support this process. Expression of human microsomal triglyceride transfer protein (MTP) and apolipoprotein B48 (apoB48) together enabled Sf-21 insect cells to secrete approximately 60-fold more lipoprotein-associated triacylglycerol than control cells. This dramatic effect demonstrates that effective partitioning of triacylglycerol into the secretory pathway requires an endoplasmic reticulum-associated neutral lipid transporter (provided by MTP) and an apolipoprotein to shuttle the lipid through the pathway. Expression of the human apoB48 gene in insect cells resulted in secretion of the protein product. Including both MTP subunits with apoB48 and oleic acid specifically increased apoB48 secretion 8-fold over individual subunits alone. To assess whether specific regions of apoB are necessary for MTP responsiveness, nine apoB segments were expressed. These included NH2-terminal segments as well as internal and COOH-terminal regions of apoB fused with a heterologous signal sequence. ApoB segments containing the NH2-terminal 17% of the protein were secreted and responded to MTP activity; however, a segment containing only the NH2-terminal 17% of the protein was not significantly responsive to MTP. Segments lacking the NH2 terminus were not MTP-responsive, and five of six of these proteins were trapped intracellularly but, in certain cases, could be rescued by fusion to apoB17. These results suggest that the NH2 terminus of apoB is necessary but not sufficient for MTP responsiveness.

Animals↗
PubMed1996

Complete nucleotide sequence of the mouse CTLA8 gene.

The gene encoding the mouse cytotoxic T-lymphocyte-associated antigen 8 (CTLA8) has been cloned and its complete nucleotide (nt) sequence determined. Sequence and polymerase chain reaction (PCR) analysis indicated that the published CTLA8 sequence[Rouvier et al., J. Immunol. 150 (1993), 5445-5456] was of rat rather than mouse origin. The mouse CTLA8 gene contains two exons and one intron. The 5'flanking region contains several consensus motifs for binding to transcription factors and the 3' untranslated region (UTR) has AU-rich motifs associated with RNA instability. The putative exon sequences predict that the full-length mouse CTLA8 molecule contains 147 amino acids (aa) and shares 88% aa identity with rat CTLA8 and 57% aa identity to HSV13, an open reading frame (ORF) from herpesvirus saimiri (HVS).

Animals↗
PubMed1996

Effect of circulating antigen on immunoscintigraphy of ovarian cancer patients using anti-CA125 monoclonal antibody.

The murine monoclonal antibody (mAb) 145-9 recognizes an epitope present on CA125 but different from the epitope defined by the mAb OC125. To evaluate the clinical usefulness of the 145-9 antibody, immunoscintigraphy was performed in ovarian cancer patients and the effect of circulating CA125 on tumor imaging was investigated. Two milligrams (74 MBq) of 111In-labeled 145-9 was injected intravenously into 11 patients with ovarian cancer. Pre-injection serum CA125 concentrations were between 166 U/ml and 7414 U/ml. Tumors were visualized in 10 of 11 patients. In two patients, lymph nodes that were not detected by other imaging modalities but were clinically suspected as metastases were visualized. There was no correlation between serum CA125 level and antibody uptake in the tumors. Immune complexes between the antibody and circulating antigen were observed in sera of all the patients, but the fraction of radioactivity in complex form did not correlate well with serum CA125 levels. The immune complexes survived in the circulation and the circulating radiolabel, including immune complexes, was still bound to solid-phase CA125. The plasma clearance rate and hepatic uptake of the antibody were not significantly affected by circulating CA125. In conclusion, the antibody 145-9 formed complexes with CA125 in vivo but this did not Compromise the outcome of antibody imaging. The antibody 145-9 can be used in immunoscintigraphy of ovarian cancer irrespective of serum CA125 level.

Adult↗
PubMed1996

In vivo inhibition of hepatitis B viral gene expression by antisense phosphorothioate oligodeoxynucleotides in athymic nude mice.

Antisense oligodeoxynucleotides strategies have been used both to study normal gene function and to block gene expression therapeutically. We have previously shown that a number of antisense oligonucleotides against hepatitis B virus (HBV) mRNA are able to inhibit viral gene expression in vitro. Here we report the establishment of an animal model producing HBV markers in athymic nude mice and inhibition of HBV gene expression and replication by antisense DNA in vivo. 2.2.15 cells (Hep-G2 cell line transfected with HBV genomes) were injected subcutaneously (s.c.) into athymic BALB/c nude mice at a total cell number of 0.5-1 x 10(8) per mouse. Transplanted tumours developed about 2 weeks after inoculation. Hepatitis B surface and e antigens (HBsAg and HBeAg), as well as HBV DNA, could be detected in the circulation of tumour-bearing mice. Hepatitis B surface antigen and hepatitis B core antigen (HBcAg) were demonstrated in tumour cells. After 10 days of tumour growth, antisense phosphorothioate oligonucleotide, complementary to the cap site of the SP II promoter of HBV mRNA, were injected by infiltration into or around the tumour as a daily dose of 20 micrograms per gram body weight. Treatment for a total of 10 days resulted in an effective inhibition of viral replication and gene expression. These results suggest therapeutic potential for antisense oligomers in the treatment of patients who are chronically infected with HBV.

Animals↗
PubMed1996

Stimulation of human lymphocyte proliferation and CD40 antigen expression by phosphorothioate oligonucleotides complementary to hepatitis B virus genome.

We have studied the proliferation and CD40 antigen expression of lymphocytes, and the cytotoxicity to monocytes, of antisense phosphorothioate oligodeoxynucleotides complementary to the SP II promoter of HBV mRNA (sequence I) and the X gene (sequence II) in patients with chronic hepatitis B. The oligo sequence I stimulated proliferation of both T and, to a lesser extent, B cells. The percentage of cells expressing CD40 in T and B cell co-cultures increased from 4.2% to 13.8% after oligo stimulation in patients, while it increased form 4.7% to 48.6% in healthy controls. The sense sequence (sequence III) of the X gene also enhanced the expression of CD40 antigen in patients with hepatitis B. The proportion of CD40 cells (26%) in a resting B-cell preparation from hepatitis B patients decreased to zero after a 5-day culture with sequence I, but IgG levels in the culture supernatant increased. The cytotoxic properties of monocytes were not influenced by the oligos. These findings indicate that antisense oligos against hepatitis B virus (HBV) have mitogenic effects on the proliferation of human lymphocytes in a non-specific manner and may activate T cells to express CD40 antigen.

Adult↗
PubMed1996

[Clinical evaluation of TPA-assay kit using monoclonal antibodies].

The clinical utility of a newly-developed radioimmunometric assay kit for tissue polypeptide antigen (TPA) was evaluated focusing on the serum diagnosis of gastrointestinal cancers. Serum TPA concentrations of normal subjects, patients with various benign and gastrointestinal malignant diseases were measured using this kit. From the TPA concentrations of normal subjects, the cut-off value was determined to be 70 U/l, which was 40 U lower than the previous kit. It showed a fairly high false positive rate in patients with liver dysfunction, so care should be taken in evaluating patients with liver disorders. It showed high positive rates in patients with wide range of gastrointestinal cancers, confirming the wide-range usefulness of this assay. Cancer patients with advanced stages and recurrences showed a higher positive rate and also higher serum TPA concentrations than in the early stages, indicating that this TPA assay is especially useful in the evaluation of therapeutic effects and in the detection of recurrent diseases.

Antibodies, Monoclonal↗
PubMed1996

[Inhibitory effect of tamoxifen on the breast cancer risk induced by exogenous estrogen in rat].

In order to investigate the inhibitory effect of tamoxifen (TAM) on breast cancer risk induced by exogenous estrogen, female rats were randomly allocated into three groups, namely DES (diethylstilbestrol) group, DES + TAM group and blank control group. We found that in DES + TAM group the levels of estradiol (62.14 pg/ml) and progesteron (15.02 ng/ml) in serum were obviously higher than those in DES group (52.69 pg/ml, 10.38 ng/ml) individually (P < 0.05, P < 0.01), the incidence (28.57%) of mammary hyperplasia was significantly lower than that in DES group (73.33%) (P < 0.025), the degree of hyperplasia markedly slighter than that in DES group (P < 0.025), and the AgNOR count (1.93%), DNA content (80.24%) and ER positive rate (36.46%) were obviously lower than those in DES group (2.43%, 95.60%, 71.71%) individually. The results indicated that TAM could increase the levels of estradiol and progesterone in serum, also could decrease the ER contents, AgNOR counts and DNA contents in mammary epithelium. It suggested that the TAM could interfere the effect on the breast carcinogenesis induced by exogenous estrogen.

Animals↗
PubMed1996

[The expression of Pax7 in C2C12 cell line and its inducible inactivation].

Pax7 is a member of mouse Pax gene family. It was expressed from day 8 to 17 p. c. during murine embryogenesis, mainly restricted to the central nerve systems. Additional Pax7 expression could be followed during myogenesis from the dermamyotome of the somites to the skeletal muscle tissues. For the first time, we demonstrated that Pax7 is expressed in mouse myoblast cell line, C2 C12. The expression of Pax7 was blocked when C2 C12 cells were induced to differentiate into myotube. Moreover, cells of mesodermal origin, in particular of the mouse embryo fibroblast cell line C3 H10 T1/2 which were especially permissive to the action of myogenic HLH proteins, did not express Pax7. These results suggest that Pax7 may be a suppressive factor for the myoblast differentiation of C2 C12 cells in vitro.

Animals↗
PubMed1996

[The mechanism of vasculogenesis: the critical role of transforming growth factor-beta 1 in the formation of vessel-like structures during the differentiation in vitro of murine embryonic stem cells].

When ES-5 cells were transfected with an exogenous porcine TGF-beta 1 gene, one can obtain clones of genetically modified ES cells with over-expression of the transfected gene. We called the genetically modified ES-5 cells as ES-T cells. When ES-T cells were used to study their differentiation in vitro by all trans-retinoic acid (RA), it was soon noticed that embryoid bodies of ES-T cells can exclusively differentiate into endothelial cells and vessel-like structures, but not in their parent ES-5 cells. The above result is the first indication that the differentiation of tubular structures in embryoid bodies of ES-T cells may somehow be related to TGF-beta 1. To demonstrate further the role of TGF-beta 1 in the formation of vessel-like structures, the cultured ES-5 cells in the presence of added rhTGF-beta 1 were closely followed in the course of their differentiation. We have, thus, demonstrated the promoting effects of exogenous rhTGF-beta 1 in the formation of vessel-like structures, morphologically similar to those structures derived from ES-T6 cells, during the differentiation of ES-5 cells, both in monolayer culture, in three dimensional collagen gel and in embryoid bodies cultured on gelatin-coated tissue culture wells. Addition of suitable amount of anti-TGF-beta 1 monoclonal antibody IgG (TB21) to the culture medium of embryoid bodies of ES-T6 cells could effectively abolish the formation of vessel-like structures induced by retinoic acid. The percentage of the inhibition was very high, giving a figure comparable to that of atypical vessel-like structures formed in the control embryoid bodies from their parent ES-5 cells. The flat epithelial-like cells and round cells differentiated from embryoid bodies of ES-T6 cells were stained rather strongly for laminin and type IV collagen by immunofluorescent procedure. The above results indicate clearly that TGF-beta 1 is a crucial factor in organizing the differentiated derivatives (endothelial-like cells and their immediate progenitor cells) from ES-T6 cells to form vessel-like structures, and that the role of TGF-beta 1 in vasculogenesis might be performed, in part, through the modulation of the composition and organization of the extracellular matrix. In addition, the enhanced expression of bFGF mRNA in derivatives differentiated from both ES-5 cells treated with rhTGF-beta 1 and ES-T6 cells were detected by Northern blot analysis. Thus, aside from its effects on extracellular matrix, TGF-beta 1 might also modulate the bioactivity of bFGF in relation to the growth of vascular endothelial cells in the present system.

Animals↗
PubMed1996

[rhTGF-beta 1 induced differentiation of human promonocytic leukemia THP-1 cells].

With promonocytic leukemia cell line THP-1 cells as an experimental material, the present paper described the proliferation, differentiation and maturation of these cells into m phi-like cells when they were treated with rhTGF-beta 1. Both cell number count and 3H-TdR uptake experiments indicated that rhTGF-beta 1 obviously inhibited the proliferation of THP-1 cells, and the inhibiting effect was related to its concentration. At the same time, the changes in the mode of cell growth and morphology occurred. The cells changed gradually from suspensive into adherent state and formed two groups of cell populations. The number of adherent cells formed was dependent on the concentration and duration of the treatment of rhTGF-beta 1. Therefore, based on the degree of inhibition of cell proliferation and the number of adherent cells with different rhTGF-beta 1 concentrations in a trial experiment, 1.25 ng/ml rhTGF-beta 1 was chosen as the dose in other experiments. From scanning electronmicroscopic observation, it was found that the external morphology of rhTGF-beta 1 treated THP-1 cells gradually transformed into typical macrophage-like cells. Concomitantly, their subcellular organelles also became progressively matured, with primary lysosomes typical for early M phi in 72 h and secondary lysosomes and phagosomes for mature M phi in 120 h of induction, as observed with transmission electron microscope. The ANAE activity, NBT reduction and phagocytosis of differentiated adherent cells were higher than those of control cells and suspensive cells. Specific anti-human TGF-beta-neutralizing mAb could completely block the differentiation of THP-1 cells into M phi-like cells. To sum up, from the results of the studies on cell morphology, growth mode, ultrastructures, phagocytosis, enzyme activation and TGF-beta 1 mAb blocking of induction and differentiation, it is clear that rhTGF-beta 1 can induce THP-1 cells to differentiate and mature into M phi-like cells, with the parallel development of cytoplasmic organoids, phenotype variation and the gaining of phagocytosis activity etc. Concordantly, rhTGF-beta 1 made the M phi-like cells to an activated state as they became matured during the induced differentiation.

Cell Transformation, Neoplastic↗
PubMed1995

Human IL-17: a novel cytokine derived from T cells.

A cDNA encoding human IL-17 (hIL-17) was cloned from a CD4+ T cell library. The predicted 155-amino acids sequence contains an N-terminal signal peptide and exhibits 72% amino acid identity with HVS13, an open reading frame from a T-lymphotropic Herpesvirus saimiri, and 63% with murine CTLA8. High levels of hIL-17 were induced from primary peripheral blood CD4+ T cells upon stimulation. When expressed in CV1/EBNA cells, recombinant hIL-17 was secreted in both glycosylated and nonglycosylated forms. A hIL-17.Fc fusion protein and supernatants from cells transfected with hIL-17 induced IL-6 and IL-8 production and enhanced the surface expression of the intracellular adhesion molecule-1 (ICAM-1) in human fibroblasts.

Amino Acid Sequence↗
PubMed1995

Elution of lipoprotein fractions containing apolipoproteins E and A-I in size exclusion on Superose 6 columns is sensitive to mobile phase pH and ionic strength.

Separation of lipoproteins secreted from McA-RH7777 (rat hepatoma) cells by Superose 6 column size-exclusion chromatography, using PBS buffer (NaCl 150 mM, sodium phosphate 10 mM, pH 7.5, EDTA 1 mM), produced apolipoprotein (apo) E or A-I profiles that did not correlate with lipoproteins separated by density ultracentrifugation. By density ultracentrifugation, apoE and apoA-I were mostly (> 90%) confined to high-density lipoproteins (HDL, d = 1.063-1.023 g/ml), but by chromatography apoE and apoA-I were recovered in all lipoprotein classes, including low-density lipoproteins (LDL), HDL, and post-HDL. Moreover, the elution volume of phenol red on Superose 6 greatly exceeded the total column volume. These discrepancies were attributable to pH and ionic strength effects. In low ionic strength, high pH buffer (Tris 25 mM, pH 8.3), elution volumes of lipoproteins, albumin, and phenol red were minimized. Elution volumes increased 25-70% when buffer pH was lowered at constant ionic strength (Tris 25 mM, pH 7.4) or when ionic strength was increased at constant pH (Tris 25 mM, pH 8.3, NaCl 500 mM). Altered phase partition appeared to cause the altered elution volumes, since recovery (measured as analyte peak area), resolution (measured as peak width at half height), and column void volume varied little from buffer to buffer. In Superose 6 size-exclusion chromatography with PBS buffer, then, elution volumes vary with pH and ionic strength. We propose that TBE buffer (Tris-borate 89 mM, pH 8.3, EDTA 2 mM) may produce fewer artefacts than PBS. With TBE there were (i) better correlation between size-exclusion and ultracentrifugal fractions, (ii) lower elution volumes, and (iii) less ¿smearing¿ of McA-RH7777 apoE and apoA-I containing lipoprotein bands.

Animals↗
PubMed1995

Ultrastructure of the spermatozoa and eggs of the ocean pout (Macrozoarces americanus L.), an internally fertilizing marine fish.

Ultrastructure of sperm and eggs of the ocean pout (Macrozoarces americanus L.), an internally fertilizing marine teleost, was examined by scanning and transmission electron microscopy. The results showed that the sperm do not have an acrosome but have a very long mid-piece (one to two times the sperm head length) containing numerous well-developed elongated mitochondria. The sperm also have two tails (is biflagellate) each consisting of nine peripheral and one central pair (9 +/- 2) of microtubules. This long mid-piece and the biflagellate nature of the sperm appear to be associated with the long life-span of the sperm and with sperm dispersal in the ovary to fertilize the eggs internally. The ocean pout eggs are enveloped by a porous chorionic membrane similar to that found in other teleosts but have two micropyles, a condition likely related to a mechanism of egg fertilization which increases the egg fertility in the presence of low sperm numbers. Following insemination, some biochemically undefined excretions appeared on the surface of fertilized eggs and led to the acquisition of adherent capability of the eggs which formed a tightly associated egg mass in sea water.

Animals↗
PubMed1995

Sequence polymorphism in the HLA-B promoter region.

Transcription of major histocompatibility complex class I genes is controlled by the class I regulatory complex in the 5' flanking region. To investigate the molecular basis of this region, we studied the polymorphism of the promoter of the HLA-B locus extending from the ATG transcription initiation signal to -284 base pairs (bp) which includes a number of cis-acting elements: interferon response sequence (IRS), enhancer A and enhancer B. Genomic DNA from 35 homozygous cell lines from the 10th International Histocompatibility Workshop and from eight heterozygous panel members was amplified using two primers designed to specifically amplify the HLA-B locus. The double-stranded polymerase chain reaction products were sequenced using the cycle sequencing technique and an ABI 373A automatic sequencer. Promoter sequences of thirty-one different HLA-B alleles were determined in this study. Within the 284 bp upstream of the ATG signal, base substitutions were observed in 23 different nucleotide positions. Our study shows a high degree of polymorphism of the HLA-B promoter region, but conserved sequences of the known cis-acting elements with the exception of enhancer B in which there are two base substitutions for B7 and B42 (position -93 and position -95). The 23 polymorphic sites can be grouped into 12 different HLA-B promoter types (groups A to M) for 31 HLA-B locus alleles. Some of the groups of alleles sharing the same promoter sequence such as, for example, group A with B51, B52, B53, and B35, might have been predicted on the basis of serological similarity and/or exon 2, 3 sequence. In other groups, such as G (B18, B37, B27), it could not have been anticipated from serological experience that B18 and B27 carry the same promoter. Several sequencing errors were detected in the HLA-B promoter sequences published previously.

Alleles↗