Variability of polyadenylation sites in mRNAs from human fetal liver

cDNA libraries of human fetal liver were constructed in pBR322 and λgt10 vectors. The libraries were screened for liver-specific clones by differential hybridization. This procedure revealed 25 and 32 liver-specific clones in plasmid and phage libraries, respectively. The majority of these clones were represented with serum albumin, fetal ^Gγ-globin and ^Aγ-globin cDNA inserts. Three types of 3′-non-coding region were found in 5 sequenced albumin cDNAs. In one type mRNA the distance between the AATAAA signal and polyadenylation site was 15 nucleotides, in 2 other types this distance was 10 and 6 nucleotides. The polyadenylation site in the ^Gγ-globin cDNA was located 2 nucleotides further from AATAAA signal, while in the ^Aγ-globin cDNA it was 2 nucleotides closer to the signal as compared with the results published previously.     

Haplotypes of nine single nucleotide polymorphisms on chromosome 19q13.2-3 associated with susceptibility of lung cancer in a Chinese population

To evaluate the joint effect of nine single nucleotide polymorphisms for three DNA repair genes in the region of chromosome 19q13.2-3 on susceptibility of lung cancer in a Chinese population, we conducted a hospital-based case–control study consisting of 247 lung cancer cases and 253 cancer-free controls matched on age, gender and ethnicity. Associations between the haplotypes and susceptibility of lung cancer were tested. The global test of haplotype association revealed a statistically significant difference in the haplotype distribution between cases and controls (global test: χ² = 60.45, d.f. = 15, P = 2.11E−07). The two haplotypes were underrepresented among cases (Hap5 defined by ERCC1118^A–ERCC2156^C–ERCC2312^G–ERCC2751^A–XRCC1194^T–XRCC1206^A–XRCC1280^G–XRCC1399^G–XRCC1632^G and Hap12 defined by ERCC1118^G–ERCC2156^C–ERCC2312^G–ERCC2751^A–XRCC1194^C–XRCC1206^A–XRCC1280^G–XRCC1399^A–XRCC1632^G). Three of the haplotypes were overrepresented among cases (Hap3 defined by ERCC1118^A–ERCC2156^C–ERCC2312^G–ERCC2751^A–XRCC1194^C–XRCC1206^A–XRCC1280^G–XRCC1399^G–XRCC1632^G, Hap4 defined by ERCC1118^A–ERCC2156^C–ERCC2312^G–ERCC2751^A–XRCC1194^C–XRCC1206^G–XRCC1280^G–XRCC1399^G–XRCC1632^A, and Hap10 defined by ERCC1118^G–ERCC2156^A–ERCC2312^G–ERCC2751^A–XRCC1194^T–XRCC1206^A–XRCC1280^G–XRCC1399^G–XRCC1632^G). Haplotypes 3 and 10 (cases = 5.7%, controls = 1.0%, OR = 6.56, 95%CI = 1.83–23.54, P = 0.001; cases = 13.3%, controls = 5.6%, OR = 2.73, 95%CI = 1.51–4.94, P = 0.0006) were the most strongly associated with increased lung cancer risk. There was considerable linkage disequilibrium exists between SNPs both within genes and between genes in the region. The two blocks for solid spine of LD and six htSNPs were found. The haplotype analysis suggested that the biologically effective polymorphisms co-segregate with some of the haplotypes. This result supports the hypothesis that the sub-region is important for lung cancer susceptibility. Haplotype studies using larger study groups will be required to obtain conclusive results.     

The position of integration affects expression of the γ-globin-encoding gene linked to HS3 in transgenic mice

Proper expression of the human β-globin (βGlb) locus is dependent on the presence of a major regulatory element located upstream from the βGlb gene cluster, the locus control region (LCR). The LCR, as well as the individual DNase-I-hypersensitive sites from which it is composed, have been shown to provide position-of-integration-independent expression in transgenic mice. Here, we report that a transgenic founder carrying multiple integrations of a hypersensitive site 3::^Aγ globin gene (HS3::^Aγ) construct produced three types of progeny, one with zero ^Aγ expression in the adult stage, one with minimal ^Aγ expression (1% of ^Aγ-expressing cells) and one with abundant ^Aγ expression (100% ^Aγ-expressing cells). The possibility that these phenotypes were due to parental imprinting or to DNA rearrangements of the transgene or to point mutations of the HS3 core or the ^Aγ promoter were excluded. The pattern of inheritance of the three HS3::^Aγ transgene phenotypes indicate that the transgene has integrated into three different chromosomes. These results provide direct evidence that the HS3 of the LCR is not sufficient to protect the ^Aγ gene from position effects excerted by the surrounding chromatin.     

Biosynthesis of Hb in individual fetal liver bursts : γ-Chain production peaks earlier than β-chain in the erythropoietic pathway

The relative synthesis of α-, β-, ^Gγ- and ^Aγ-globin chains has been evaluated in single fetal liver bursts, which were grown in methylcellulose cultures, individually labelled with [³H]leucine and then analysed via iso-electric focusing. Well-hemoglobinized bursts demonstrate a homogeneous globin synthetic pattern, characterized by prevalent HbF (+some HbA) synthesis: thus, they apparently originate from a homogeneously programmed population of erythroid burst-forming unit (BFU-E). On day 8–9 of culture, the synthetic pattern in ‘mature’ (i.e., well-hemoglobinized) bursts has been compared with that in simultaneously-grown, ‘immature’ (i.e., poorly-hemoglobinized) colonies. These patterns have been further compared with that in ‘matured’ bursts (identified in situ as immature on day 8–9 and labelled 2–4 days later when matured). The ‘immature’ colonies showed very low levels of relative β-globin synthesis, while the ‘mature’ ones demonstrated a more elevated production of β-chain. Significantly, the ‘matured’ bursts showed a globin chain synthetic pattern similar to that of previously labelled ‘matured’ colonies. It is postulated therefore that in fetal liver (and also in adult marrow) the synthesis of γ-chain is linked to an early differentiation stage of erythroblasts, while β-globin synthesis is largely activated at a more advanced maturation stage.     

Analyses of In Vitro Nonenzymatic Glycation of Normal and Variant Hemoglobins by MALDI-TOF Mass Spectrometry

MALDI-TOF mass spectrometry is used here to differentiate different glycoisoforms of normal and variant hemoglobins (Hbs) in nonenzymatic in vitro glycation. Single, double, and/or multiple glycation of the α-globin, β-globin, and/or γ-globin is observed. Different glycation rates are observed for various Hbs, and the normal Hb A has the slowest rate. Although the Hb A is relatively stable upon condensation with glucose at 37°C, the variants Hb C, Hb E, Hb F, Hb Leiden, and Hb San Diego are less stable. In addition, data reveal that the number of glucose attached/Hb molecule (state of glycation) increases with longer incubation time, higher glucose concentration, and higher temperature. The pH dependence of the state of glycation is more complex and varies for different Hbs. Although pH has little effect on the state of glycation for Hb C, Hb E, and Hb Leiden, it increases for Hb A and Hb F upon changing the pH of the solution from phosphate buffer saline (pH 7.4) to carbonate buffer (pH 10). Results obtained in this study could lead to the inference that the linkage of Hbs with glucose occurs in diabetic conditions in vivo (37°C, ∼neutral pH, ∼0.007 M glucose), and the state of glycation is more severe in the individuals who carry abnormal Hbs.     

A comparison of hemoglobins in five species of Microtus

The composition of tryptic peptides was determined for Hb (major or fast electrophoretic component) from four additional species of Microtus; M. p. pennsylvanicus, M. abbreviatus, M. miurus, and M. oeconomus, The amino acids from the four Hb were compared with Hb from M. p. tananaensis, and, on the basis of the combination of analyses for the several Hb. Ca 95% of the overall amino acid composition was considered. The compositions of most of the homologous peptides were identical, and two deletions in the sequence of 21 amino acids β 41–61 are believed to characterize the Hb of all four species. From differences in peptide composition the following substitutions were inferred. In the β chain, M. pennsylvanicus (2 ssp) differed from other species at two positions, 8: Ser vs Thr and 12: Thr vs Ash. In the β chain M. pennsylvanicus (2 ssp) differed from other species at two positions, β45: Phe vs Leu, and β50: Ser-Glx vs Thr. M. p. pennsylvanicus differed from M. p. tananaensis at position β88: Val-Leu vs Leu. All species showed ambiguity among the amino acids Ala-Ser-Phe-Leu centred presumably in positions β129 and β130. On the basis of an incomplete examination, the slow electrophoretic component of M. abbreviatus Hb could not be seen to differ from the fast component in its peptide map or in the general composition of its and β peptides.     

Identification of five hemoglobins in B6C3F1 mice by mass spectrometry and sequence analysis

The aim of the work is to identify and characterize the hemoglobins found in B6C3F1 mice using mass spectrometry. The primary structures are compared to those reported for BALB/c mice. Individual hemoglobin chains were isolated by reversed-phase high performance liquid chromatography (RP-HPLC). The molecular masses of the globins were determined using electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI). The purified globin chains were enzymatically cleaved and the resulting peptides were separated by RP-HPLC. The chains were identified by N-terminal sequencing and mass spectrometry (MALDI). Selected peptides were analysed by Edman degradation. ESI analysis indicates that B6C3F1 mice have two -globin chains (-1 and -2) and at least three β-globin chains, β-1, β-2 and β-3. This is one additional - and one additional β-globin chain than reported in the literature for BALB/c mice. Mass and sequence analysis of enzymatically generated peptides showed variations in the amino acid sequence in the -1, -2, β-2 and β-3 chains compared to the BALB/c mouse hemoglobins (, β^minor and β^major). The study showed that mass spectrometry in combination with traditional protein chemistry is able to identify and locate minor protein sequence variations.     

A Large Cohort of Hemoglobin Variants in Thailand: Molecular Epidemiological Study and Diagnostic Consideration

Background

Hemoglobin (Hb) variants are structurally inherited changes of globin chains. Accurate diagnoses of these variants are important for planning of appropriate management and genetic counseling. Since no epidemiological study has been conducted before, we have investigated frequencies, molecular and hematological features of Hb variants found in a large cohort of Thai subjects.

Materials and Methods

Study was conducted on 26,013 unrelated subjects, inhabiting in all geographical parts of Thailand over a period of 11 years from January 2002-December 2012. Hb analysis was done on high performance liquid chromatography (HPLC) or capillary electrophoresis (CE). Mutations causing Hb variants were identified using PCR and related techniques.

Results

Among 26,013 subjects investigated, 636 (2.4%) were found to carry Hb variants. Of these 636 subjects, 142 (22.4%) carried α-chain variants with 13 different mutations. The remaining included 451 (70.9%) cases with 16 β-chain variants, 37 (5.8%) cases with Hb Lepore (δβ-hybrid Hb) and 6 (0.9%) cases with a single δ-chain variant. The most common α-globin chain variant was the Hb Q-Thailand (α^{74GAC-CAC, Asp-His}) which was found in 101 cases (15.8%). For β-globin chain variants, Hb Hope (β^{136GGT-GAT, Gly-Asp}) and Hb Tak (β^{146+AC, Ter-Thr}) are the two most common ones, found in 121 (19.0%) and 90 (14.2%) cases, respectively. Seven Hb variants have never been found in Thai population. Hb analysis profiles on HPLC or CE of these variants were illustrated to guide presumptive diagnostics.

Conclusions

Hb variants are common and heterogeneous in Thai population. With varieties of thalassemias and hemoglobinopathies in the population, interactions between them leading to complex syndromes are common and render their diagnoses difficult in routine practices. Knowledge of the spectrum, molecular basis, genotype-phenotype correlation and diagnostic features should prove useful for prevention and control of the diseases in the region.     

Endothelial nitric oxide synthase gene haplotypes and circulating nitric oxide levels significantly associate with risk of essential hypertension

Nitric oxide (NO), a potent vasodilator, plays a pivotal role in blood pressure regulation. Endothelial NO synthase gene (NOS3) polymorphisms influence NO levels. Here, we investigated the role of the – 922A/G, – 786T/C, 4b/4a, and 894G/T polymorphisms of the NOS3 and NO_x levels in 800 consecutive unrelated subjects comprising 455 patients of essential hypertension and 345 controls. The polymorphisms were investigated independently and as haplotypes. Plasma NO_x levels (nitrate and nitrite) were estimated by the Griess method. Genotype frequencies for the –786T/C, 4b/4a, and 894G/T polymorphisms differed significantly (P < 0.001) between patients and controls and were associated with an increased risk of hypertension (OR = 2.0, OR = 3.8, OR = 1.6, respectively). The 4-locus haplotypes ATaG (H1), ATaT (H2), and GCaG (H3) were significantly associated with essential hypertension and served as susceptible haplotypes (P ≤ 0.0001). On the other hand, haplotypes ATbG (H4) and GTbG (H5) were negatively associated with hypertension and served as protective haplotypes (P < 0.0001). NO_x levels were significantly lower in patients than controls (P < 0.0001). The individual polymorphisms showed marginal association with NO_x level; however, the susceptible haplotype H2 associated significantly with lower NO_x levels in patients (P < 0.001) and conversely the haplotype H4 with higher NO_x levels in controls (P < 0.001). In conclusion, the 4b/4a and likely – 786T/C polymorphisms were identified as the determinants modifying the risk of hypertension. This study identifies the NOS3 variants and haplotypes as genetic risk factors and as useful markers of increased susceptibility to the risk of essential hypertension.     

Prosomatostatin is proteolytically processed at the amino terminal segment by subtilase SKI-1

Processing of prohormones to generate active products typically occurs at basic residues via cleavage by proprotein convertases. A less common type of cleavage is mediated at hydrophobic (L, V, F, N) or small amino acid (A, T, S) residues. Efforts to identify the proteinases responsible for processing precursors at their hydrophobic amino acids has led to the recent cloning of a new type-1 membrane-bound subtilase called SKI-1. The NH₂-terminal region of prosomatostatin, previously shown to contain a sorting signal for the regulated secretory pathways, is processed to generate PSST_[1–10]. The exact cleavage mechanism is unknown, but has been assumed to involve monobasic processing at Lys¹³ followed by carboxypeptidase trimming. We found that K13A mutation did not block PSST_[1–10] production. Since the prosomatostatin sequence R⁸–Q⁹–F¹⁰–L¹¹↓ qualifies as a potential SKI-1 substrate, using a vaccinia virus expression system along with HPLC and radioimmunoassays, we observed that overexpression of recombinant SKI-1 in COS-1 and HEK-293 cells significantly increased the production of PSST_[1–10]. Additionally, in CHO cells lacking SKI-1, there was a significant reduction in PSST_[1–10] production which could be increased upon SKI-1 stimulation. Mutagenesis studies showed that efficient processing of PSST to PSST_[1–10] required the RXRXXL motif. However, this NH₂-terminal cleavage was not a prerequisite for the formation of SST-14 and SST-28.     

Sensitive detection of human globin chains by microbore high-performance liquid chromatography and its forensic application

This paper describes a simple and sensitive high-performance liquid chromatographic (HPLC) method for the detection of human globin chains in blood and bloodstains. The method involves direct injection of the filtered samples of dilute hemolysates or bloodstain extracts onto a microbore C₄ reversed-phase column (2.1 mm I.D.) with UV detection at 220 nm. Microbore HPLC offers a significant improvement in sensitivity with little loss of the resolution of globin chains and only small variations in the determination of γ chain composition. The detection limit of hemoglobin (Hb) was 0.1 μg, which is equivalent to about 1 nl of fresh whole blood. Umbilical cord blood could be differentiated from adult blood in stains that were up to twenty weeks old, by the presence of γ globin chains. The present method will be useful for detection of abnormal Hbs and for the determination of γ chain composition in clinical laboratories, as well as in the practice of forensic science for the analysis of minute amounts of blood and bloodstains.     

Biosynthesis of globin chains in fetal liver and adult marrow cultures : Comparative analysis of individual colonies derived from early, intermediate or late erythroid progenitors

The relative synthesis of globin chains (α,β,^Gγ,^Aγ) has been comparatively evaluated in erythroid colonies from 26 fetal livers (7–15 gestational week) and 13 ‘normal’ adult marrows. Clusters deriving from erythroid colony-forming units (CFU-E) were analysed either individually or in pools of –20 colonies. Bursts deriving from earlier erythroid progenitors (erythroid burst-forming unit, ‘primitive’ or ‘mature’, P-BFU-E or M-BFU-E, respectively) were always analysed individually. Since γ-globin synthesis peaks earlier than β-chain production in both the fetal and the adult erythroblastic pathway, the globin synthetic pattern has been comparatively evaluated, in so far as possible, in colonies at an homogenous, advanced stage of hemoglobinization.In fetal liver cultures, the relative β-synthesis in CFU-E clusters, M- and P-BFU-E bursts constantly shows low, fairly uniform values. In adult marrow cultures, the relative γ-production in the corresponding three classes of colonies is characterized by low, rather homogeneous levels (except for more elevated γ-synthetic values occasionally observed in pooled CFU-E clusters comprising a majority of poorly-hemoglobinized colonies). A gradual decrease of relative γ-production has never been observed in colonies deriving from progressively more differentiated erythroid progenitors of both fetal and adult origin.These results suggest that fetal and adult BFU-E are endowed respectively with a program for prevailing HbF or HbA synthesis, which is not substantially modulated at the level of erythroid progenitors under standard culture conditions. By implication, it is postulated that, in fetal and more particularly adult age, modulation of globin synthesis is mediated via mechanism(s) acting at the level of erythroblasts, i.e. at the level of the early γ- and the late β-synthesis in their maturation pathway. The Hb switch (i.e. the switch from prevailingly HbF to HbA synthesis program) is possibly dependent on the ontogenic ‘maturation’ of BFU-E (and/or stem cells), which peaks in the perinatal period.     

Limitations in the use of 3-hydroxy fatty acid analysis to determine endotoxin in mammalian samples

3-Hydroxy fatty acids (3-OH FAs) of 10–18-carbon chain lengths are constituents of the lipopolysaccharide of Gram-negative bacteria. These acids are used as chemical markers for determining endotoxin in environmental samples. The present communication addresses the question whether this type of analysis also would be applicable to mammalian samples. Low levels (6.1±1.6–94.0±23.2 pmol/ml) of the studied 3-OH FAs were detected in blood from both conventional and germ-free rats. The levels were considerably higher (0.0–1.06±0.17 nmol/mg) in livers. The amounts of the 3-OH FAs did not differ between the two groups of rats. All analyses were made by gas chromatography–tandem mass spectrometry (GC–MSMS) for unequivocal identification. The results illustrate a limitation in using 3-OH FA analysis to determine endotoxin in mammalian samples since these acids may represent not only endotoxin but also products from mammalian mitochondrial fatty acid β-oxidation.     

Hetero-polynuclear complexes containing bridging diphenylphosphino-alkenes and -alkynes. The crystal structure of an Rh2{μ−ν:ν−P(Ph2) (CH2)3C≡CR}Co2 complex

The phosphinoalkenes Ph₂P(CH₂)_nCH=CH₂ (n= 1, 2, 3) and phosphinoalkynes Ph₂P(CH₂)_n C≡CR (R = H, N = 2, 3; R = CH₃, N = 1) have been prepared and reacted with the dirhodium complex (η−C₅H₅)₂Rh₂(μ−CO) (μ−η²−CF₃C₂CF₃). Six new complexes of the type (ν−C₅H₅)₂(Rh₂(CO) (μ−η¹:η¹−CF₃C₂CF₃)L, where L is a P-coordinated phosphinoalkene, or phosphinoalkyne have been isolated and fully characterized; the carbonyl and phosphine ligands are predominantly trans on the Rh---Rh bond, but there is spectroscopic evidence that a small amount of the cis-isomer is formed also. Treatment of the dirhodium-phosphinoalkene complexes with (η−CH₃C₅H₄)Mn(CO)₂thf resulted in coordination of the manganese to the alkene function. The Rh₂---Mn complex [(η−C₅H₅)₂Rh₂(CO) (μ−η¹:η¹−CF₃C₂CF₃) {Ph₂P(CH₂)₃CH=CH₂} (η−CH₃C₅H₄)Mn(CO)₂] was fully characterized. Simi treatment of the dirhodium-phosphinoalkyne complexes with Co₂(CO)₈ resulted in the coordination of Co₂(CO)₆ to the alkyne function. The Rh₂---Co₂ complex [(η−C₅H₅)₂Rh₂(CO) (μ−η¹:η¹−CF₃C₂CF₃) {Ph₂PCH₂C≡CCH₃}Co₂(CO)₂], C₃₇H₂₅Co₂F₆O₇PRh₂, was fully characteriz spectroscopically, and the molecular structure of this complex was determined by a single crystal X-ray diffraction study. It is triclinic, space group (C_i¹, No. 2) with a = 18.454(6), B = 11.418(3), C = 10.124(3) Å, = 112.16(2), β = 102.34(3), γ = 91.62(3)°, Z = 2. Conventional R on |F| was 0.052 fo observed (I > 3σ(I)) reflections. The Rh₂ and Co₂ parts of the molecule are distinct, the carbonyl and phosphine are mutually trans on the Rh---Rh bond, and the orientations of the alkynes are parallel for Rh₂ and perpendicular for Co₂. Attempts to induce Rh₂Co₂ cluster formation were unsuccessful.     

Intrinsic potential for high fetal hemoglobin production in a Druze family with β-thalassemia is due to an unlinked genetic determinant

Summary The mechanism for elevated production of fetal hemoglobin (Hb F) in a Druze patient with °-thalassemia intermedia was investigated. Heterozygous family members exhibited normal Hb F levels, suggesting that the increase in -gene expression in the propositus may be partly due to anemic stress. Erythroid progenitors of these family members cultured in vitro [burst forming units (erythroid); (BFUe)] showed elevated synthesis of Hb F, indicating the existence of a genetically determined intrinsic capacity for high Hb F production in this family. The propositus was found to be homozygous for a IVS2-position 1 mutation, on the background of Mediterranean haplotype I, which is not known to be linked to high Hb F production. Moreover, extensive molecular studies of the -globin gene cluster, including sequence analysis of the promoter regions of the -globin genes, did not reveal any cisacting mechanism that could account for the high Hb F production in the propositus. A young niece of the propositus with °-thalassemia major was recently discovered, who was homozygous for the same -globin allele and haplotype as the propositus. However, unlike her uncle, she does not have a high Hb F level and presents with a severe clinical course. Her inability to produce high Hb F suggests that the genetic determinant for increased -gene expression in the propositus is unlinked to the -globin gene cluster.     

Deletions within the Mouse β-Globin Locus Control Region Preferentially Reduce β Globin Gene Expression

The mouse β-globin gene cluster is regulated, at least in part, by a locus control region (LCR) composed of several developmentally stable DNase I hypersensitive sites located upstream of the genes. In this report, we examine the level of expression of the β^min and β^maj genes in adult mice in which HS2, HS3, or HS5,6 has been either deleted or replaced by a selectable marker via homologous recombination in ES cells. Primer extension analysis of RNA extracted from circulating reticulocytes and HPLC analysis of globin chains from peripheral red blood cells revealed that all mutations that reduce the overall output of the locus preferentially decrease β^min expression over β^maj. The implications of these findings for the mechanism by which the LCR controls expression of the β^maj and β^min promoters are discussed.