Adult Reticulocytes Research Articles

Two 3' sequences direct adult erythroid-specific expression of human beta-globin genes in transgenic mice.

Previous experiments have demonstrated that the human beta-globin gene is correctly regulated in transgenic mice. The beta-globin gene is not expressed in yolk sac-derived erythroid cells in early embryonic development but is expressed concomitantly with the adult mouse beta-globin genes in 14- to 16-day fetal liver and adult reticulocytes. In an attempt to localize sequences that direct erythroid-specific expression, fragments of the human beta-globin gene were inserted in the opposite orientation 200 base pairs (bp) upstream of an intact human A gamma marker gene, which is not expressed on its own in mouse fetal liver. In the experiments reported here, two beta-globin 3' sequences activated the marker gene specifically in fetal liver. One sequence is located in a 250-bp Pst I fragment 550-800 bp downstream from the poly(A) site; the other is located near an EcoRI site in the third exon. These two sequences are active individually, and their combined effect is greater than their effects alone. beta-Globin 5' sequences from -815 to -50 were also analyzed for activity in this assay. The 5' sequences did not activate the marker gene when tested alone but did stimulate expression that was already directed to adult erythroid tissue by the two 3' sequences. These results suggest that three separate sequences are involved in human beta-globin gene regulation. The two 3' sequences act as adult erythroid enhancers and the 5' sequence stimulates expression that is already determined to be erythroid specific.

Independent expression of the two mouse adult ?-globin genes

Dot blot hybridization was used to determine the relative amounts of the beta-major and beta-minor globin RNAs present in reticulocytes of mice at 14.5 and 17.5 days of gestation, newborns, and adults of the Hbab/Hbab;Hbbs2/Hbbs2 globin genotype. RNAs isolated from embryonic yolk sac, fetal liver, and adult reticulocytes were hybridized with the following labeled DNA probes: alpha-1, beta-minor specific, and beta-major specific. The level of beta-sminor RNA in reticulocytes at 14.5 and 17.5 days of gestation is nearly the same as in induced reticulocytes of adult mice. In contrast, the level of beta-s2major RNA in reticulocytes at 14.5 days of gestation is 0.23 X and at 17.5 days of gestation is 0.66 X the amount found in reticulocytes of adult mice. These results correlate well with earlier observations that the beta-sminor globin gene approaches its normal adult level of expression by 14.5 days of gestation, whereas the beta-s2major globin gene expression increases between 14.5 days of gestation and 6 days postnatally. They indicate that the differential expression of beta-sminor and beta-s2major globins during development is regulated at the level of transcription. Expression of the beta-minor globin gene in reticulocytes of adult normal mice is not maximal, however, because the levels of the beta-minor globin and its RNA are increased further in reticulocytes of thalassemic mice.

Polyadenylation of the Xenopus beta 1 globin mRNA at a downstream minor site in the absence of the major site and utilization of an AAUACA polyadenylation signal.

We show that adult reticulocytes of Xenopus laevis produce two forms of the beta 1 globin mRNA that differ in their site of polyadenylation. The minor site of polyadenylation is located 46 nucleotides downstream of the major site and is used in approximately 1% of mRNA molecules. A fusion gene was constructed containing the promoter from the thymidine kinase gene of herpes simplex virus fused to the protein coding, 3'-noncoding and 3'-flanking sequences of the X. laevis beta 1 globin gene. When injected into the nuclei of Xenopus oocytes, transcripts of this fusion gene were accurately and efficiently spliced and polyadenylated. The proportion of fusion gene transcripts terminating at the major and minor polyadenylation sites after injection into oocytes was approximately similar to that found in reticulocytes. When the AATAAA sequence element upstream from the major site was deleted, the minor site was used with a high (greater than 90%) efficiency. Therefore, by comparing the ratio of polyadenylation at the major and minor sites, it is possible to determine the effect of sequence alterations at the major site. In a construct where the AATAAA polyadenylation signal was changed to AATACA a high proportion (35%) of transcripts continued to be polyadenylated at the major site. This suggests a surprisingly high degree of flexibility in the precise polyadenylation signal.

Isolation of non-globin genes expressed preferentially in mouse erythroid cells.

Genomic DNA recombinants were isolated from a library of Balb-C mouse genomic DNA fragments cloned in lambda Ch4A by screening with cDNA derived from 13d foetal liver cell or adult reticulocyte poly A+ RNA. Subsequent screening enabled us to identify non-globin genomic sequences whose expression appeared exclusive to or elevated in erythroid cells. Further analysis of the structure and expression of these sequences was performed using Southern blot and DNA or RNA dot hybridisation analysis. In one recombinant part of the cloned genomic sequence corresponded to an erythroblast specific mRNA identified previously by Affara et al, (5).

The aminoacyl-tRNA population of human reticulocytes.

The aminoacyl-tRNA population of human reticulocytes has been examined. These studies include: 1) determination of the levels of amino acid acceptance for 20 aminoacyl-tRNAs; 2) comparison of 20 aminoacyl-tRNAs from human reticulocytes to those of rabbit reticulocytes by reverse phase chromatography; 3) comparison of the levels of Ile- and Leu-tRNAs in fetal and adult reticulocytes; and 4) determination of the codon recognition properties of human Ala-, Asn-, His-, Leu-, Thr-, and Val-tRNAs. These studies provide evidence that the aminoacyl-tRNA population of human reticulocytes is adapted to the requirements of protein synthesis.

The asynchrony of γ- and β-chain synthesis during human erythroid cell maturation: III. γ- and β-mRNA in immature and mature erythroid clones

Globin chain synthesis and the accumulation of globin mRNAs were examined in immature and mature erythroid clones produced by circulating erythroid progenitors (burst forming units) of the neonate. Immature clones were composed of early erythroblasts containing little hemoglobin while the mature clones appeared at later days in culture and were composed of well hemoglobinized erythroblasts. There was a strikingly higher γ γ + β chain synthetic ratio in the nonhemoglobinized clones compared to their fully hemoglobinized counterparts. RNA was prepared from immature and from mature colonies and α-, β-, and γ-mRNA content was measured by solution hybridization to α-, β-, and γ-[ 32P]DNA probes. The probes were sense strand coding globin sequences prepared from chimeric plasmids by isolation of nick-translated [ 32P]DNA which annealed to cord blood or adult reticulocyte globin mRNA. In three experiments, the γ γ + β mRNA ratios in immature bursts were 0.58, 0.55, and 0.70 while ratios in the mature counterparts were 0.21, 0.38, and 0.45. There was excellent agreement between globin chain synthetic ratios and globin mRNA ratios suggesting that the globin mRNA species present in immature and mature erythroblasts are translated with similar efficiencies. These data suggest that the maturational modulation of non-α-globin chain synthesis is not under translational control. The absolute mRNA values (molecules per cell) consistently showed similar amounts of γ-mRNA per cell in both the immature and the mature cell populations. The most striking effect of maturation was the increase in the amount of β-mRNA per cell (and the concomitant increase in α-mRNA). These data, together with previously published results on globin chain biosynthesis suggest that the maturation related decline in γ/γ + β chain ratios is due to asynchrony in γ- and β-mRNA accumulation. Apparently γ-mRNA accumulates early (at or near the onset of globin synthesis) and the accumulation of β-mRNA follows. This asynchrony in mRNA accumulation results in a decline in the relative synthesis of fetal hemoglobin during maturation.

The duplicated human α-globin genes: Their relative expression as measured by RNA analysis

The human α-globin genes are duplicated and encode identical polypeptides. Recently we detected in cloned genomic DNAs characteristic sequence differences between the 3′ untranslated regions of the 5′ (α2) and 3′ (α1) genes, not previously recognized by direct analysis of mRNA and cDNA transcripts. Based on these untranslated region differences, we have now used S1 nuclease mapping of RNA to detect and quantitate the two predicted α-mRNA species. With this assay we have examined the relative expression of the α-globin genes during normal development and in α-thalassemia syndromes. In normal adult reticulocytes, α2 RNA is slightly more abundant than the α1 species (ratio 60:40). This relative abundance of the a RNAs was consistently observed in fetal blood and liver RNA samples from 10 weeks of gestation to birth. In both deletion and nondeletion forms of α thalassemia, only the α1 RNA species was present in erythroid RNA. These studies resolve prior conflicts regarding the heterogeneity of α RNA and establish the normal pattern of relative α-gene expression during development independent of protein variants. RNA analysis also permits for the first time identification of the mutant genes in nondeletion forms of a thalassemia.

Cloning of a new mouse foetal β-globin mRNA sequence

A novel globin cDNA recombinant (pFG5) has been isolated from a 14-15 day Porton mouse foetal liver cDNA library. It codes for a beta-like globin mRNA expressed in foetal liver-derived erythroblasts and erythrocytes but not in adult reticulocytes nor in yolk sac derived nucleated erythrocytes. It is also found in Friend cells induced to differentiate by DMSO. The nucleotide sequence of pFG5 confirms that it does not code for the beta major or beta minor globin chains nor the embryonic epsilon Y2 globin chain; but it is identical to the published partial sequence of the epsilon Y3 globin gene over the region of overlap (78 nucleotides).

Sequence differences between mouse embryonic y- and adult beta-globin messenger ribonucleic acids.

Poly(A)-containing RNA was isolated from yolk sac cells of BDF1 hybrid mice on the 12th day of gestation. In vitro translation of the poly(A)-containing RNA by a messenger RNA (mRNA) dependent reticulocyte lysate system revealed that this RNA contained a high concentration of mRNAs from two embryonic globin chain subunits, y-1 and y-2. Cross hybridization of complementary DNA (cDNA) to the embryonic globin mRNAs with adult reticulocyte globin mRNAs and with cloned cDNAs for adult alpha- and beta-globin chain mRNAs showed that the sequence of embryonic globin y-chain mRNA differed from that of adult beta-chain mRNA.

Unequal alpha and beta globin mRNA in reticulocytes of normal and mutant f/f fetal mice.

Messenger RNA (mRNA) was isolated from adult as well as normal and mutant f/f fetal mouse reticulocytes by poly-U affinity chromatography. mRNA from normal adult reticulocytes directed equal synthesis of alpha and beta globin chains in wheat germ cell free translational system. mRNA from either normal or mutant f/f fetal reticulocytes directed 40% more alpha globin synthesis than beta globin synthesis. These observations are suggestive that there is significantly more alpha mRNA than beta mRNA in normal and mutant f/f fetal mouse reticulocytes.