Tissue stem cells and cancer stem cells: potential implications for gastric cancer.

Gastric cancer remains the second leading cause of death in the world today, making the search for its molecular and cellular basis an important priority. Though recognition of the tight link between inflammation and tumorigenesis is centuries old, only recently are the pieces of the etiological puzzle beginning to fall together. Recent advances in gastric stem cell biology appear to be central to this slowly resolving puzzle. At least two types of stem cells may be important. Resident adult or tissue stem cells may, in a chronically inflamed environment, slowly acquire a series of genetic and epigenetic changes that lead to their emergence as ''cancer stem cells''. This scenario has not yet been proven experimentally, although the first step, prospective recognition of a gastric stem cell has recently been conquered. Alternatively, the setting of chronic inflammatory stress and injury may lead to loss of the indigenous gastric stem cells from their niches; bone marrow derived stem cells may then be recruited to and engraft into the gastric epithelium. Such recruited cells have the potential to contribute to the tumor mass. Indeed, evidence supporting this scenario has been published. Here, we review these recent findings and discuss implications for the future.

Genes encoding pancreatic polypeptide and neuropeptide Y are on human chromosomes 17 and 7.

Pancreatic polypeptide and neuropeptide Y share 50% amino acid homology (18 out of 36 residues), suggesting that they may have common ancestral origins. cDNA clones complementary to human mRNAs encoding pancreatic polypeptide and neuropeptide Y were used to detect specific human genomic DNA sequences in human-mouse somatic cell hybrid lines. The pancreatic polypeptide gene (PPY) segregated with human chromosome 17, while the neuropeptide Y gene (NPY) segregated with human chromosome 7. Examination of cell hybrids with chromosomal rearrangements assigned PPY to the p11.1-qter region and NPY to the pter-q22 region of their respective chromosomes.

Methylation-enhanced binding of Sp1 to the stage selector element of the human gamma-globin gene promoter may regulate development specificity of expression.

The human gamma-globin gene promoter contains a stage selector element (SSE) responsible for preferential interaction of the promoter with a powerful erythroid-specific enhancer in the fetal developmental stage (S.M. Jane, P.A. Ney, E.F. Vanin, D.L. Gumucio, and A.W. Nienhuis. EMBO J. 11:2691-2699, 1992). The element binds two proteins, the ubiquitous activator Sp1 and a protein previously known as -50 gamma and now named the stage selector protein (SSP). Binding of the second protein correlates with SSE activity in transient-transfection assays. We now report that a de novo binding site for the SSP is created by the -202(C-->G) mutation that causes hereditary persistence of fetal hemoglobin (HPFH). This site functions in an analogous manner to the SSE in hybrid beta-promoter/reporter gene constructs transfected into K562 cells. In contrast, the wild-type -202 sequence, which fails to bind the SSP, is incapable of activating the beta-gene promoter. Both the -50 and -202 HPFH sites for SSP binding overlap a consensus sequence for the transcriptional regulator Sp1. In addition, both sites contain CpG dinucleotides that are contact bases for SSP. Since the gamma promoter is known to be hypomethylated in fetal cells but fully methylated at CpG residues in adult erythroid cells, we examined the effects of this DNA modification on protein binding to the two regions. Gel mobility shift assays with nuclear extract from K562 cells (which contain both Sp1 and SSP) demonstrate preferential binding of SSP to the SSE and HPFH sites under conditions in which probe was limiting. Methylation of the CpG residues reverses this preference only in the SSE site, with a marked increase in the binding of Sp1 at the expense of the SSP. Purified Sp1 binds with 10-fold higher affinity to the methylated than to the nonmethylated -50 probe but with the same affinity to the -202 HPFH probe. The methylation-induced preferential binding of Sp1 to the SSE at the expense of SSP may be part of the mechanism by which the gamma genes are repressed in normal adult erythroid cells. In cells containing the -202 HPFH mutation, the inability of Sp1 to displace SSP in the methylated state may explain the persistence of gamma-promoter activity and gamma-gene expression observed in adults with this mutation.

Nuclear proteins that bind the human gamma-globin gene promoter: alterations in binding produced by point mutations associated with hereditary persistence of fetal hemoglobin.

The molecular mechanisms responsible for the human fetal-to-adult hemoglobin switch have not yet been elucidated. Point mutations identified in the promoter regions of gamma-globin genes from individuals with nondeletion hereditary persistence of fetal hemoglobin (HPFH) may mark cis-acting sequences important for this switch, and the trans-acting factors which interact with these sequences may be integral parts in the puzzle of gamma-globin gene regulation. We have used gel retardation and footprinting strategies to define nuclear proteins which bind to the normal gamma-globin promoter and to determine the effect of HPFH mutations on the binding of a subset of these proteins. We have identified five proteins in human erythroleukemia cells (K562 and HEL) which bind to the proximal promoter region of the normal gamma-globin gene. One factor, gamma CAAT, binds the duplicated CCAAT box sequences; the -117 HPFH mutation increases the affinity of interaction between gamma CAAT and its cognate site. Two proteins, gamma CAC1 and gamma CAC2, bind the CACCC sequence. These proteins require divalent cations for binding. The -175 HPFH mutation interferes with the binding of a fourth protein, gamma OBP, which binds an octamer sequence (ATGCAAAT) in the normal gamma-globin promoter. The HPFH phenotype of the -175 mutation indicates that the octamer-binding protein may play a negative regulatory role in this setting. A fifth protein, EF gamma a, binds to sequences which overlap the octamer-binding site. The erythroid-specific distribution of EF gamma a and its close approximation to an apparent repressor-binding site suggest that it may be important in gamma-globin regulation.

Concerted evolution of human amylase genes.

Cosmid clones containing 250 kilobases of genomic DNA from the human amylase gene cluster have been isolated. These clones contain seven distinct amylase genes which appear to comprise the complete multigene family. By sequence comparison with the cDNAs, we have identified two pancreatic amylase genes and three salivary amylase genes. Two truncated pseudogenes were also recovered. Intergenic distances of 17 to 22 kilobases separate the amylase gene copies. Within the past 10 million years, duplications, gene conversions, and unequal crossover events have resulted in a very high level of sequence similarity among human amylase gene copies. To identify sequence elements involved in tissue-specific expression and hormonal regulation, the promoter regions of the human amylase genes were sequenced and compared with those of the corresponding mouse genes. The promoters of the human and mouse pancreatic amylase genes are highly homologous between nucleotide -160 and the cap site. Two sequence elements thought to influence pancreas-specific expression of the rodent genes are present in the human genes. In contrast, similarity in the 5' flanking sequences of the salivary amylase genes is limited to several short sequence elements whose positions and orientations differ in the two species. Some of these sequence elements are also associated with other parotid-specific genes and may be involved in their tissue-specific expression. A glucocorticoid response element and a general enhancer element are closely associated in several of the amylase promoters.

Simultaneous expression of salivary and pancreatic amylase genes in cultured mouse hepatoma cells.

The tissue-specific expression of two types of mouse amylase genes does not overlap in vivo; the Amy-1 locus is transcribed in the parotid gland and the liver, while expression of Amy-2 is limited to the pancreas. We identified a mouse hepatoma cell line, Hepa 1-6, in which both amylase genes can be simultaneously expressed. Amy-1 is constitutively active in these cells and is inducible by dexamethasone at the level of mRNA. We demonstrated that the liver-specific promoter of Amy-1 is utilized by the dexamethasone-treated hepatoma cells, and that glucocorticoid consensus sequences are present upstream of this promoter. Amy-2 is not detectable constitutively, but can be activated if the cells are cultured in serum-free medium containing dexamethasone. Expression of Amy-2 in a nonpancreatic cell type has not previously been observed. We speculate that induction of Amy-1 and activation of Amy-2 may involve different regulatory mechanisms. Hepa 1-6 cells provide an experimental system for molecular analysis of these events.

Phylogenetic footprinting reveals a nuclear protein which binds to silencer sequences in the human gamma and epsilon globin genes.

Tissue- and developmental stage-specific expression of the human beta-like globin genes is regulated by a combination of ubiquitous and erythroid-restricted trans factors that bind to cis elements near each of the five active genes. Additional interactions of these cis and trans factors with sequences located in the far 5' end of the cluster occur by as yet obscure mechanisms. Because of the complexity of this regulatory puzzle, precise identification of the determinants that control hemoglobin switching has proven difficult. Phylogenetic footprinting is an evolutionary approach to this problem which is based on the supposition that the basic mechanisms of switching are conserved throughout mammalian phylogeny. Alignment of the 5' flanking regions of the gamma genes of several species allows the identification of footprints of 100% conserved sequence. We have now tested oligomers spanning 13 such phylogenetic footprints and find that 12 are bound by nuclear proteins. One conserved element located at -1086 from the gamma genes exhibits repressor activity in transient transfection studies. The protein that binds this element, CSBP-1 (conserved sequence-binding protein 1), also binds at three sites within a silencer element upstream from the epsilon globin gene. Further analysis reveals that the CSBP-1 binding activity is identical to that of a recently cloned zinc finger protein that has been shown to act as a repressor in other systems. The binding of CSPB-1 to silencer sequences in the epsilon and gamma globin genes may be important in the stage-specific silencing of these genes.

Interstrain variation in amylase gene copy number and mRNA abundance in three mouse tissues.

Amylase expression in strain YBR differs in several respects from the standard mouse phenotype. The synthesis of salivary amylase is elevated twofold in YBR mice and the synthesis of pancreatic amylase is reduced to one-half the normal rate. We have compared the concentrations of amylase mRNA in the parotid, liver and pancreas of YBR mice with those in strains A/J and C3H. We observed differences in amylase mRNA abundance which can account for the levels of amylase protein synthesis in the parotid and pancreas of these strains. Unexpectedly, the concentration of amylase mRNA in the liver of YBR mice was also higher than in the other strains. Since liver amylase is transcribed from the same gene as parotid amylase, duplication of the Amy-1 locus could account for the elevated mRNA concentration in both tissues. Quantitative analysis of genomic DNA by Southern blotting provided direct evidence for duplication of Amy-1 in strain YBR.

Dynamics of regulatory evolution in primate beta-globin gene clusters: cis-mediated acquisition of simian gamma fetal expression patterns.

Phylogenetic reconstructions by parsimony were carried out on an enlarged body of primate gamma1 and gamma2-globin sequences. The results confirm that gamma1 and gamma2 arose from a tandem duplication in an ancient simian lineage ancestral to both platyrrhines (New World monkeys) and catarrhines (Old World monkeys and hominoids). Gene conversions between the two gamma homologs were frequent over the gamma gene proper but less frequent over the 5' flanking and very infrequent over the 3' flanking regions. The ancient platyrrhine conversion in the most distal 5' flanking region had the polarity of gamma2-->gamma1. Recent platyrrhine conversions between 5' regulatory sequences were very infrequent, in striking contrast to catarrhines which have large, uninterrupted stretches of converted 5' regulatory sequences. Comparisons of reconstructed ancestral primate and simian gamma promoter sequences revealed an accumulation of 21 nucleotide substitutions concentrated in or near cis-elements that may have mediated the change from embryonic to fetal gamma expression. Almost all 21 substitutions were retained in the lineages leading to functional gamma genes of extant catarrhines (both gamma1 and gamma2) and platyrrhines (most often gamma2). Fewer of these simian specific substitutions were retained in the platyrrhine gamma1 genes and new mutations occurred more often in the platyrrhine gamma1 than gamma2 promoters.

Locus control regions of mammalian beta-globin gene clusters: combining phylogenetic analyses and experimental results to gain functional insights.

Locus control regions (LCRs) are cis-acting DNA segments needed for activation of an entire locus or gene cluster. They are operationally defined as DNA sequences needed to achieve a high level of gene expression regardless of the position of integration in transgenic mice or stably transfected cells. This review brings together the large amount of DNA sequence data from the beta-globin LCR with the vast amount of functional data obtained through the use of biochemical, cellular and transgenic experimental systems. Alignment of orthologous LCR sequences from five mammalian species locates numerous conserved regions, including previously identified cis-acting elements within the cores of nuclease hypersensitive sites (HSs) as well as conserved regions located between the HS cores. The distribution of these conserved sequences, combined with the effects of LCR fragments utilized in expression studies, shows that important sites are more widely distributed in the LCR than previously anticipated, especially in and around HS2 and HS3. We propose that the HS cores plus HS flanking DNAs comprise a 'unit' to which proteins bind and form an optimally functional structure. Multiple HS units (at least three: HS2, HS3 and HS4 cores plus flanking DNAs) together establish a chromatin structure that allows the proper developmental regulation of genes within the cluster.

Fire and ICE: the role of pyrin domain-containing proteins in inflammation and apoptosis.

The genetic bases for several human autoinflammatory syndromes have recently been identified, and the mutated proteins responsible for these diseases are rapidly being characterized. Here, we examine two of these newly identified proteins, pyrin (also called marenostrin, product of the familial Mediterranean fever locus, MEFV) and cryopyrin (product of the CAIS1 locus, and mutated in familial cold urticaria, Muckle Wells syndrome and chronic infantile neurological cutaneous and articular syndrome). Both pyrin and cryopyrin contain an N-terminal domain that encodes a death domain-related structure, now known as the pyrin domain, or PyD. We trace the molecular interactions mediated by these PyDs, examine the evolution of the family of molecules containing this domain, and discuss the function of PyD-containing proteins and their homologues. Synthesis of the available data indicates that both pyrin and cryopyrin interact via their PyDs with a common adaptor protein, ASC. ASC itself participates in at least three important cellular processes: apoptosis, recruitment and activation of pro-caspase-1 (with associated processing and secretion of IL-1beta), and activation of NF-kappaB (a transcription factor involved in both initiation and resolution of the inflammatory response). Through PyD:PyD interactions, pyrin and cryopyrin, as well as several related, but still uncharacterized PyD containing proteins, appear to modulate the activity of all three of these processes, each of which plays a crucial role in the inflammatory pathways that characterize the innate immune system.

Peroxisome proliferator activated receptor gamma in colonic epithelial cells protects against experimental inflammatory bowel disease.

INTRODUCTION: Peroxisome proliferator activated receptor gamma (PPARgamma) is expressed in epithelial cells, macrophage, and T and B lymphocytes. Ligand induced activation of PPARgamma was reported to attenuate colitis activity but it is not clear whether this protection is mediated by epithelial or leucocyte PPARgamma. METHODS: Mice with targeted disruption of the PPARgamma gene in intestinal epithelial cells, generated using a villin-Cre transgene and floxed PPARgamma allele and designated PPARgamma(DeltaIEpC), were compared with littermate mice having only the PPARgamma floxed allele with no Cre transgene that expressed PPARgamma in the gut, designated PPARgamma(F/F). Colitis was induced by administering dextran sodium sulphate (DSS) and the two mouse lines compared for typical symptoms of disease and expression of inflammatory cytokines. RESULTS: PPARgamma(DeltaIEpC) mice displayed reduced expression of the PPARgamma target genes ADRP and FABP in the gut but were otherwise normal. Increased susceptibility to DSS induced colitis, as defined by body weight loss, colon length, diarrhoea, bleeding score, and altered histology, was found in PPARgamma(DeltaIEpC) mice in comparison with PPARgamma(F/F) mice. Interleukin (IL)-6, IL-1beta, and tumour necrosis factor alpha mRNA levels in colons of PPARgamma(DeltaIEpC) mice treated with DSS were higher than in similarly treated PPARgamma(F/F) mice. The PPARgamma ligand rosiglitazone decreased the severity of DSS induced colitis and suppressed cytokine production in both PPARgamma(F/F) and PPARgamma(DeltaIEpC) mice. CONCLUSIONS: These studies reveal that PPARgamma expressed in the colonic epithelium has an endogenous role in protection against DSS induced colitis and that rosiglitazone may act through a PPARgamma independent pathway to suppress inflammation.

Isolation of intestinal epithelial cells for the study of differential gene expression along the crypt-villus axis.

Methods for the differential isolation of intestinal epithelial cells from crypt and villus compartments of small intestine have been used in the study of intestinal functions. However, the use of different methods has resulted in discrepant conclusions as to the localization of expressed genes. Therefore, we undertook a careful comparison of two methods of intestinal epithelial cell isolation, the distended intestinal sac method and the everted intestinal sac method. The isolated cell fractions (distended sac fractions 1-10, everted sac fractions 1-5) were evaluated for the expression of two mRNAs whose localization along the crypt-villus axis had been previously elucidated by in situ hybridization: cytochrome P-450IIB1 (expressed in villus cells) and cryptdin (expressed in crypt cells). Northern blots of total or poly(A)+ RNA from each cell population showed that the first fractions from both methods contained P-450IIB1 mRNA, suggesting that both methods allowed the isolation of cells originating from the villus. Cryptdin mRNA was detected only in cell fractions 5-10 using the distended sac method and was not detected in any fractions from the everted sac method. [3H]thymidine incorporation demonstrated that dividing (crypt) cells were successfully removed by the distended sac method, but remained with the everted sac intestinal remnant. Finally, light and electron microscopy of the isolated cells as well as the intestinal remnants confirmed that while undifferentiated crypt cells were present in distended sac cell fractions 9 and 10, they remained with the everted sac remnant. Thus the distended sac protocol was useful for the isolation of cells from tip and crypt compartments.(ABSTRACT TRUNCATED AT 250 WORDS)

Granulocyte-macrophage colony-stimulating factor-dependent growth and erythropoietin-induced differentiation of a human cell line MB-02.

Peripheral blood blasts from a patient with acute megakaryoblastic leukemia were placed into liquid cultures with recombinant growth factors. Growth, but not differentiation, was supported by interleukin-3 (IL-3) or granulocyte-macrophage colony-stimulating factor (GM-CSF) for the first 30 days of culture. Sustained growth occurred only with GM-CSF and gave rise to the cell line MB-02, which has been in continuous culture for over 1 year. The cell line retained the surface phenotype of the leukemic megakaryoblasts except for the loss of glycoproteins Ib and IIb/IIIa, which were induced after exposure to phorbol esters. The induction of erythropoiesis occurred when GM-CSF-deprived cells were cultured with erythropoietin (Epo). Well-defined morphologic stages of differentiation ranging from primitive erythroblasts to nuclei-extruding normoblasts were seen. Transforming growth factor-beta inhibited GM-CSF- and Epo-dependent growth, but not erythroid maturation. Indirect immunofluorescence using globin chain-specific monoclonal antibodies detected fetal, but not adult hemoglobin in the uninduced cells. beta-globin was induced and gamma-globin was increased after Epo exposure. Both globin species accumulated in the developing erythrocytes until terminal differentiation. Quantitative S1 analysis of beta-like globin transcripts showed very low levels of epsilon- and beta-globin expression and high levels of gamma-globin expression in cells maintained in GM-CSF. Five days after induction with Epo, epsilon message decreased to barely detectable levels while gamma and beta transcripts increased threefold and 20-fold, respectively. This novel cell line not only retains many characteristics of the leukemic megakaryoblasts from which it was derived, but also can be induced to recapitulate apparent normal erythropoiesis.

Expression of the human amylase genes: recent origin of a salivary amylase promoter from an actin pseudogene.

The human genes encoding salivary amylase (AMY1) and pancreatic amylase (AMY2) are nearly identical in structure and sequence. We have used ribonuclease protection studies to identify the functional gene copies in this multigene family. Riboprobes derived from each gene were hybridized to RNA from human pancreas, parotid and liver. The sizes of the protected fragments demonstrated that both pancreatic genes are expressed in pancreas. One of the pancreatic genes, AMY2B, is also transcribed at a low level in liver, but not from the promoter used in pancreas. AMY1 transcripts were detected in parotid, but not in pancreas or liver. Unexpected fragments protected by liver RNA led to the discovery that the 5' regions of the five human amylase genes contain a processed gamma-actin pseudogene. The promoter and start site for transcription of AMY1 are recently derived from the 3' untranslated region of gamma-actin. In addition, insertion of an endogenous retrovirus has interrupted the gamma-actin pseudogene in four of the five amylase genes.

Hepatocytes of Zones 1 and 3 conjugate sulfobromophthalein with glutathione.

The capacity of hepatocytes of Zones 1 and 3 of the liver acinus to conjugate sulfobromophthalein (BSP) with glutathione and to secrete the conjugate into bile was studied. BSP was infused into the in situ perfused rat liver in concentrations of 0.01 and 0.05 mM. Perfusions were performed either in the portal to hepatic vein direction (forward perfusion) or in the hepatic vein to portal vein direction (retrograde perfusion). Hepatocytes contributing to the uptake, metabolism and biliary secretion of BSP were directly assessed qualitatively by light microscopy, and also semiquantitatively by microspectrophotometry. BSP was taken up predominantly by hepatocytes of Zone 1 during forward perfusion and by those of Zone 3 during the retrograde perfusion of BSP. The biliary products of BSP metabolism by each acinar zone were subsequently assessed. Hepatocytes of both Zones 1 and 3 of the acinus secreted BSP into bile in the form of BSP-glutathione, and BSP-glutathione conjugate represented about 78% of the total BSP secreted into bile by each zone. The rate of BSP biliary secretion by both zones was similar at a concentration of 0.01 mM BSP, while a slight decrease (15%) in BSP secretory rate by hepatocytes of zone 3 was observed at concentrations of BSP near biliary Tm. Liver perfusion with exogenous BSP-glutathione provided results similar to those obtained with BSP. In contrast, the perfusion of 3,6- dibromosulphthalein , a compound which is not conjugated by hepatocytes, resulted in similar biliary secretory rates regardless of the direction of perfusion. These results indicate that: (a) the capacity for glutathione conjugation of BSP is distributed among hepatocytes of all acinar zones; (b) near biliary Tm, the biliary secretory rate of BSP by hepatocytes of Zone 3 is slower than that of hepatocytes of zone 1, and (c) in vivo, all hepatocytes likely contribute to the uptake, metabolism and biliary secretion of BSP.

cDNA cloning, chromosomal localization and evolutionary analysis of mouse vacuolar ATPase subunit D, Atp6m.

The multi-subunit vacuolar ATPase pump uses ATP hydrolysis to move protons into membrane bound compartments. The pump is involved in a variety of cellular functions, including regulation of cytosolic pH, vesicular transport, endocytosis, secretion, and apoptosis. Here, we describe the cDNA cloning and chromosomal mapping of subunit D of murine V-ATPase. The mouse gene, designated Atp6m, maps to Chromosome 12, in a region of high homology with human chromosome 14q24. Evolutionary analysis of subunit D orthologs in a variety of other species reveals that this is a highly conserved protein that has been under remarkably strong negative selection during evolution, most likely reflecting its critical role in multiple cellular processes.

Phylogenetic footprinting reveals unexpected complexity in trans factor binding upstream from the epsilon-globin gene.

The human epsilon-globin gene undergoes dramatic changes in transcriptional activity during development, but the molecular factors that control its high expression in the embryo and its complete repression at 6-8 weeks of gestation are unknown. Although a putative silencer has been identified, the action of this silencer appears to be necessary but not sufficient for complete repression of epsilon gene expression, suggesting that multiple control elements may be required. Phylogenetic footprinting is a strategy that uses evolution to aid in the elucidation of these multiple control points. The strategy is based on the observation that the characteristic developmental expression pattern of the epsilon gene is conserved in all placental mammals. By aligning epsilon genomic sequences (from -2.0 kb upstream to the epsilon polyadenylylation signal), conserved sequence elements that are likely binding sites for trans factors can be identified against the background of neutral DNA. Twenty-one such conserved elements (phylogenetic footprints) were found upstream of the epsilon gene. Oligonucleotides spanning these conserved elements were used in a gel-shift assay to reveal 47 nuclear binding sites. Among these were 8 binding sites for YY1 (yin and yang 1), a protein with dual (activator or repressor) activity; 5 binding sites for the putative stage selector protein, SSP; and 7 binding sites for an as yet unidentified protein. The large number of high-affinity interactions detected in this analysis further supports the notion that the epsilon gene is regulated by multiple redundant elements.

Targeted disruption of the mouse villin gene does not impair the morphogenesis of microvilli.

The small intestine is functionally dependent on the presence of the brush border, a tightly packed array of microvilli that forms the amplified apical surface of absorptive cells. In the core of each microvillus, actin filaments are bundled by two proteins, villin and fimbrin. Previous in vitro studies using antisense approaches indicated that villin plays an important role in the morphogenesis of microvilli. To examine the in vivo consequences of villin deficiency, we disrupted the mouse villin gene by targeted recombination in mouse embryonic stem cells. A beta-galactosidase cDNA was also introduced into the villin locus by the targeting event. Homozygous villin-deficient mice are viable, fertile, and display no gross abnormalities. Intact microvilli are present in the small intestine, colon, kidney proximal tubules, and liver bile canaliculi. Although subtle ultrastructural abnormalities can be detected in the actin cores of small intestinal microvilli, localization of sucrase isomaltase, brush border myosin I, and zonula occludens I to the microvillar surface of the small intestine is normal. Thus, in vivo, villin plays a minor or redundant role in the generation of microvilli in multiple absorptive tissues.

Evolution of a fetal expression pattern via cis changes near the gamma globin gene.

One basis for the evolution of organisms is the acquisition of new temporal and spatial domains of gene expression. Such novel expression domains could be generated either by cis sequence changes that alter the complement of trans-acting regulators binding to control elements or by changes in the expression patterns of one or more of the regulatory (trans) factors themselves. The gamma globin gene is a prime example of a gene that has undergone a distinct change in temporal expression at a defined time in evolution. Approximately 35-55 million years ago, the previously embryonic gamma gene acquired a fetal expression pattern. This change occurred in a simian primate ancestor after the separation of simian and prosimian primates but before the further separation of the major simian lineages; thus, the (prosimian) galago gamma gene retains the ancestral embryonic expression pattern, whereas the (simian) human gamma gene is fetal. This analysis of galago and human gamma genes in transgenic mice demonstrates that cis changes in sequences within a 4.0-kilobase region surrounding the gamma gene were responsible for the evolution of a novel fetal expression pattern in the gamma globin genes of simian primates.