Human gamma-globin genes silenced independently of other genes in the beta-globin locus.

Erythropoiesis during human development is characterized by switches in expression of beta-like globin genes during the transition from the embryonic through fetal to adult stages. Activation and high-level expression of the genes is directed by the locus control region (LCR), located 5' to the epsilon gene. The location of the LCR and its role in directing high-level expression of the globin genes has led to the suggestion that competition from the beta gene for interaction with the LCR has a major role in silencing the fetal gamma genes during adult life. We have now constructed lines of transgenic mice containing the human A gamma globin gene linked to the LCR. We observe high-level expression of the transgene in the embryonic stages but silencing of the gene in adult animals. We conclude that the gamma gene is not deregulated by the presence of the LCR and that competition from the beta gene is not required for silencing of the gamma genes in adult life. The silencing is therefore likely to be mediated by stage-specific factors binding to sequences immediately flanking the genes.     

Structure of the human fetal globin gene locus.

We have derived a 'map' of restriction enzyme sites in and around the human gamma-globin genes. This has enabled us to show that there are two gamma-globin genes per haploid set, that the genes contain 'introns' within the same regions of DNA as the human beta and delta-globin genes, and that the genes are 3,500 base pairs apart. We conclude that the correct gene organisation of the human beta-like globin locus is GgammaAgammadeltabeta.     

Developmental regulation of human fetal-to-adult globin gene switching in transgenic mice

Transgenic mice containing a human fetal (gamma-) or adult (beta-) globin gene linked to the beta-globin gene locus activation region (LAR) express the gene throughout development. By contrast, transgenic mice containing LAR linked to both a fetal and an adult globin gene display the normal developmental switch from fetal to adult gene expression. This suggests that the human fetal-to-adult globin gene switch is controlled through a mutually exclusive interaction between LAR and either the gamma- or beta-globin gene, resulting in the expression of only one gene at any given moment.     

Site-independent expression of the chicken beta A-globin gene in transgenic mice

The level of expression of exogenous genes carried by transgenic mice typically varies from mouse to mouse and can be quite low. This behaviour is attributed to the influence of the mouse chromatin near the site of transgene integration. This 'position effect' has been seen in transgenic mice carrying the human beta-globin gene. It was however, abolished when DNase I hypersensitive sites (normally found 65 to 44 kilobases (kb) upstream) were linked to the human beta-globin transgene. Thus, the upstream DNA (previously named a dominant control or locus activation region, now denoted a locus control region) conferred the ability to express human beta-globin at high levels dependent on copy number on every mouse carrying the construct. We report here an investigation of chicken beta A-globin gene expression in transgenic mice. A 4.5-kb fragment carrying the beta A-globin gene and its downstream enhancer, without any far upstream elements, is sufficient to ensure that every transgenic mouse expresses chicken globin messenger RNA at levels proportional to the transgene copy number. Thus the chicken DNA elements that allow position-independent expression can function in mice. In marked contrast to the human beta cluster, these elements are no farther than 2 kb from the gene. The location of the elements within the cluster demonstrates that position independence can be mediated by DNA that does not define a gene cluster boundary.     

Developmental regulation of a cloned adult beta-globin gene in transgenic mice

At different stages of mammalian development, distinct embryonic, fetal and adult haemoglobins are synthesized in erythroid cells, a process termed haemoglobin switching. The cellular and molecular mechanisms controlling haemoglobin switching have been intensively studied, but remain poorly understood. To study the developmental regulation of globin gene expression, we have produced transgenic mice in which cloned globin genes are present in erythroid cells throughout development. Recently, we reported that adult mice in several transgenic lines carrying a hybrid mouse/human adult beta-globin gene, expressed the gene in a correct tissue-specific manner. This finding raised the question of whether an exogenous globin gene could also be subject to appropriate stage-specific regulation. We report here that the hybrid beta-globin gene, like the endogenous adult beta-globin genes, is inactive in yolk sac-derived embryonic erythroid cells and is expressed for the first time in fetal liver erythroid cells. Our results indicate that a stage-specific pattern of expression can be conferred by cis-acting regulatory elements closely linked to an adult beta-globin gene. They also suggest that the embryonic and adult beta-globin genes in the mouse are activated (or repressed) by distinct trans-acting regulatory factors present in embryonic, fetal and adult erythroid cells.     

植物转基因沉默的原因及对策

转基因植物中转基因沉默已成为植物基因工程的一大障碍,转基因沉默的原因是多方面的,可能是由于转录前外源基因和内源基因的结构特性、伴置效应以及宿主植物的遗传调控;也可能是因为转录时启动子、转录因子和终止子的作用;还可能是由于转录后的修饰作用、外源基因表达特异性和环境等因素。为了克服转基因植物中转基因沉默,可以采取下列对策：选择适宜的外源基因和调控元件、采用适当的转化方法、使用更加简便快捷的筛选策略等。     

An embryonic pattern of expression of a human fetal globin gene in transgenic mice

During the evolution of the beta-globin family gene in vertebrates, different globin genes acquired different developmental patterns of expression. In mammals, specific 'embryonic' beta-like globins are synthesized in the earliest erythroid cells, which differentiate in the yolk sac of the embryo. In most mammals the embryonic globin chains are replaced by 'adult' beta-globins in fetal and adult erythrocytes, which arise in the liver and bone marrow, respectively. However, in simian primates (including humans), a distinct 'fetal' type of beta-like globin chain predominates in fetal erythroid cells. Based on the pattern of DNA sequence homologies between different mammalian species, these fetal globin genes, G gamma and A gamma, are thought to have descended from an ancestral gene, 'proto-gamma', which was embryonic in its pattern of expression. In the mouse, as well as in most other mammalian species, the descendants of the proto-gamma gene continue to function as embryonic genes. To investigate the evolutionary changes that led to the 'fetal recruitment' of the gamma-globin genes in primates, we have introduced the cloned human G gamma-globin gene into the mouse germ line. We report here that the human G gamma gene reverts to an embryonic pattern of expression in the developing mouse. This observation suggests that during evolution a shift occurred in the timing of expression of a trans-acting signal controlling the proto-gamma gene.     

Specific expression of a foreign beta-globin gene in erythroid cells of transgenic mice

The globin gene family represents an attractive system for the study of gene regulation during mammalian development, as its expression is subject to both tissue-specific and temporal regulation. While many aspects of globin gene structure and expression have been described extensively, relatively little is known about the cis-acting DNA sequences involved in the developmental regulation of globin gene expression. To begin to experimentally define these regulatory sequences, we have taken the approach of introducing cloned globin genes into the mouse germ line and examining their expression in the resulting transgenic animals. Here we describe a series of transgenic mice carrying a hybrid mouse/human adult beta-globin gene, several of which express the gene exclusively or predominantly in erythroid tissues. These studies demonstrate that regulatory sequences closely linked to the beta-globin gene are sufficient to specify a correct pattern of tissue-specific expression in a developing mouse, when the gene is integrated at a subset of foreign chromosomal positions.     

Selective activation of human beta-but not gamma-globin gene in human fibroblast x mouse erythroleukaemia cell hybrids.

The human alpha- and beta-globin genes have been activated in MEL X human fibroblast cell hybrids. However, even though the human gamma- and beta-globin genes are closely linked and were shown in these hybrid clones to be present in approximately equal numbers, no human gamma-globin mRNA was produced. Thus, the human beta- and gamma-globin genes in these cells are differentially regulated apparently by a positive regulatory factor(s) specific for individual globin genes.     

Sequence organization and genomic complexity of primate theta 1 globin gene, a novel alpha-globin-like gene

The alpha-like and beta-like globin genes have provided a paradigm for the study of molecular evolution and regulation of multigene families in eukaryotes. The human alpha-globin gene cluster, which is on chromosome 16 (ref. 1), consists of six genes arranged in the order 5'-zeta(embryonic)-psi zeta-psi alpha 2-psi alpha 1-alpha 2(adult)-alpha 1(adult)-3'. DNA sequencing data have demonstrated that zeta (ref. 6) and alpha 2 (or alpha 1, refs 7-9) are the embryonic and adult genes, respectively, while psi zeta (ref. 6), psi alpha 2 (ref. 5) psi alpha 1 (ref. 10) are all inactive pseudogenes. Restriction mapping analysis has shown that the structure of this locus in several anthropoid primates is nearly identical to that of the human. Recently, we have isolated the adult alpha-globin gene region from orang-utan, olive baboon and rhesus macaque by molecular cloning. We report here the complete nucleotide sequence of a gene located immediately downstream from the adult alpha 1-globin gene of the orang-utan, along with its flanking DNA. We designate this gene as theta 1, and show that it contains the essential sequence elements required for an expressive gene. The putative polypeptide is 141 amino acids long, identical to that of the alpha- or zeta-globin, but its predicted amino-acid sequence is nearly as different from the orang-utan alpha-globin (55 differences) as the human zeta-globin is from the human alpha-globin (59 differences), suggesting an ancient history for the theta 1-globin gene. Results of blot hybridization experiments using the cloned orang-utan theta 1 gene sequence as probe demonstrate a similar alpha 2-alpha 1-theta 1 linkage map existing in the human genome. Furthermore, multiple copies of sequences homologous to the theta 1 gene are detected in both human and orang-utan. These results cast a new light on the primate alpha-globin gene family, and have intriguing implications for the existence of previously unreported, functional globin-like gene(s) in the primate genomes.     

Interchromosomal associations between alternatively expressed loci

The T-helper-cell 1 and 2 (T(H)1 and T(H)2) pathways, defined by cytokines interferon-gamma (IFN-gamma) and interleukin-4 (IL-4), respectively, comprise two alternative CD4+ T-cell fates, with functional consequences for the host immune system. These cytokine genes are encoded on different chromosomes. The recently described T(H)2 locus control region (LCR) coordinately regulates the T(H)2 cytokine genes by participating in a complex between the LCR and promoters of the cytokine genes Il4, Il5 and Il13. Although they are spread over 120 kilobases, these elements are closely juxtaposed in the nucleus in a poised chromatin conformation. In addition to these intrachromosomal interactions, we now describe interchromosomal interactions between the promoter region of the IFN-gamma gene on chromosome 10 and the regulatory regions of the T(H)2 cytokine locus on chromosome 11. DNase I hypersensitive sites that comprise the T(H)2 LCR developmentally regulate these interchromosomal interactions. Furthermore, there seems to be a cell-type-specific dynamic interaction between interacting chromatin partners whereby interchromosomal interactions are apparently lost in favour of intrachromosomal ones upon gene activation. Thus, we provide an example of eukaryotic genes located on separate chromosomes associating physically in the nucleus via interactions that may have a function in coordinating gene expression.     

The gene for theta-globin is transcribed in human fetal erythroid tissues

A new gene like the alpha-globin gene has been identified in higher primates at the 3' end of the alpha-globin gene cluster. There is some controversy as to whether this gene, theta, is a functional globin gene or a non-functional pseudogene. The high degree of sequence conservation displayed by theta between primates and various mammals, such as horse and rabbit, suggests that this gene is functional in some species. Furthermore, theta encodes a 141-amino-acid polypeptide in sequence similar to alpha-globin and appears to possess functional RNA-processing signals. But the promoter region of theta is unlike the other globin genes because its CCAAT and ATA box sequences are displaced from the coding sequence by the insertion of a 200-base-pair GC-rich sequence. We demonstrate here the presence of theta-globin messenger RNA in human fetal erythroid tissue, but not in adult erythroid or other non-erythroid tissues. Furthermore, theta-globin mRNA is detectable in significant amounts in a human erythroleukaemic cell line. These results predict that theta-globin protein will be found in the early stages of human fetal development. Surprisingly, the promoter sequence of theta-globin does not correspond to the CCAAT and ATA box sequences of the gene but rather lies within the adjacent GC-rich sequence, resulting in a heterogeneous series of mRNA 5' ends 50-10 base pairs to 5' of the initiation codon. This type of promoter is reminiscent of that found in housekeeping genes such as adenine deaminase and hypoxanthine-guanine phosphoribosyl-transferase.     

A haemoglobin switching activity modulates hereditary persistence of fetal haemoglobin

During human development there is a switch from fetal to adult haemoglobin formation, reflecting the differential expression of fetal (G gamma and A gamma) and adult (beta and delta) globin genes. Mutations that inhibit this switch produce variants of the syndrome of hereditary persistence of fetal haemoglobin (HPFH). Adult heterozygotes for these mutants produce 15-30% fetal haemoglobin (HbF) in their red cells. The general assumption is that the mutations result in a permanent switching on of gamma-globin genes. Here, however, we show that fetal globin expression can be turned off in cultures of HPFH cells by an uncharacterized factor in fetal sheep serum. This is the first demonstration that mutations affecting the developmental expression of globin genes can be modulated by exogenous factors. The findings raise the possibility that the phenotype of HPFH is not simply the direct result of mutations in or around globin genes but the consequence of the mutations on the interaction of globin genes with trans-acting regulatory factors.     

Rearrangement of a chicken immunoglobulin gene occurs in the lymphoid lineage of transgenic mice

Immunoglobulin (Ig) and T-cell antigen receptor genes rearrange through identical heptamer-nonamer recognition sequences during entry of cells into the B or T lymphoid lineage. A similar enzymatic machinery may be used to perform these highly cell-specific events in these two types of lymphoid cells. We have investigated what the signal may be that triggers the rearrangement of one or other of the receptor genes in B or T cells. Mice from three transgenic lines carrying two, four or twenty copies of the unrearranged chicken lambda light-chain locus were analysed. In all three lines the chicken Ig transgene rearranges in B cells; in the line with 20 copies, a rearranged fragment can also be detected in thymus DNA. We conclude that the inserted chicken light-chain locus in its natural configuration contains target sequences that permit specific rearrangement in mouse lymphoid B cells, but that this precise differentiation step may be deregulated in thymic cells when the physiological level of relevant information is experimentally altered.