Evolution of a Fetal Expression Pattern via cis Changes near the gamma  Globin Gene*

(Received for publication, January 31, 1997, and in revised form, March 20, 1997)

Catherine TomHon Dagger , Wei Zhu Dagger , David Millinoff Dagger , Kenji Hayasaka Dagger , Jerry L. Slightom §, Morris Goodman and Deborah L. Gumucio Dagger par

From the Dagger  University of Michigan, Department of Anatomy and Cell Biology, Ann Arbor, Michigan 48109-0616, § Pharmacia and Upjohn Company, Molecular Biology Unit 7242, Kalamazoo, Michigan 49007 and the  Department of Anatomy, Wayne State School of Medicine, Detroit, Michigan 48201

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

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.


INTRODUCTION

Reconstruction of the evolutionary history of the mammalian beta  globin gene cluster indicates that in the common ancestor of marsupial and placental mammals (135 MYA),1 two beta -like globin genes existed, each with a different temporal expression pattern (1). This two gene cluster, 5'-epsilon -beta -3', persists in present day marsupials; the epsilon  gene is expressed in embryonic life, whereas the beta  gene is active postembryonically (1, 2). In early placental mammals, however, prior to the mammalian radiation (80-100 MYA), a beta  duplication produced two postembryonic genes (delta  and beta ), and epsilon  duplications produced three embryonic genes (epsilon , gamma , and eta ) (3, 4). Although gene duplications, gene inactivations, gene deletions, and even whole locus duplications or triplications have further modified the beta  globin loci of all present day eutherian mammals, a clear relationship to this ancestral five-member cluster can still be appreciated (3). Moreover, these five genes have, for the most part, retained their ancestral programs of stage specificity; the epsilon  gene is embryonic, and the beta  gene is postembryonic in all extant eutherian mammals. However, some lineage-specific alterations in the temporal expression of globin genes have occurred. An important example is the gamma  globin gene; originally embryonic in its expression pattern, the gamma  gene was recently recruited to be a fetal gene in anthropoid (simian) primates (5).

All simian gamma  genes studied to date are expressed fetally, whereas prosimian gamma  genes retain the embryonic pattern characteristic of other (nonprimate) eutherian mammals (4-7). Therefore, acquisition of fetal specificity can be traced to a relatively narrow evolutionary window (35-55 MYA), after the separation of simian (catarrhine and platyrrhine) from prosimian (galagos and lemurs) primates but prior to the divergence of platyrrhine (New World Monkeys) from catarrhine (Old World Monkeys, apes, and human) primates (4, 5). Two other events can be traced to this same evolutionary window: duplication of the gamma  gene and a burst of base substitutions that occurred both in the promoter region of the gamma  globin gene and in coding regions (5). Amino acid substitutions resulting from the coding region changes led to loss of 2,3-diphosphoglycerate binding ability, resulting in a fetal hemoglobin molecule that could bind oxygen with increased affinity and thus facilitate the transfer of oxygen from mother to fetus. Both the promoter and coding region base substitutions were subsequently fixed during further evolution of platyrrhine and catarrhine primates. This pattern of accelerated base substitution followed by decelerated rates of substitutions in the same regions has been considered indicative of the spreading and subsequent preservation of adaptive substitutions (8-10).

Although these three molecular events (fetal recruitment, gene duplication, and the burst of promoter substitutions) cannot be temporally ordered based on current phylogenetic evidence, one possible scenario is that the duplication of the gamma  gene provided a redundant substrate for the accumulation of base changes that altered gamma  stage specificity (6, 11). Once such a base change was collected, perhaps in the duplicate gamma  gene at greater distance from the LCR (Agamma or gamma 2), they may have been selected for and subsequently transferred to the other gene (Ggamma or gamma 1) by gene conversion. Indeed, evidence exists for gene conversions of this polarity (11, 12). Implicit in this hypothetical scenario is the assumption that cis mechanisms were responsible for the fetal recruitment of the gamma  gene, an assumption that has not been definitively tested. To examine this question directly, transgenic mice were generated in which expression of the galago gamma  (embryonic) and the human gamma  (fetal) genes could be compared. Analysis of the stage-specific expression of these two gamma  genes in the mice reveals distinctly different patterns; galago gamma  gene expression is embryonic and is silenced in the fetal liver, whereas human gamma  gene activity peaks in fetal life. cis differences in the gamma  gene fragments must therefore direct these different expression patterns.


EXPERIMENTAL PROCEDURES

DNA Fragments Used in Transgene Construction

The galago gamma  fragment used spans sequences 10508-14995 of GenBankTM entry M73981[GenBank] of the Galago crassicaudatis beta  globin cluster. The human gamma  fragment corresponds to sequences 38084-42140 of the human globin cluster (GenBankTM HUMHBB) and includes the Agamma 3' enhancer region (13). The galago gamma  gene contains several small insertions not present in the human gene, accounting for the slight size difference in these two fragments (4057 and 4487 bp for the human and galago gamma  gene, respectively). Human epsilon  sequences and HS3 sequences used for both constructs correspond to HUMHBB coordinates 3267-5172 (HS3) and 17841-21241 (epsilon ).

Transgenic Mice

Insert DNA was purified away from vector sequences prior to injection of the constructs for production of transgenic mice. Purified DNA fragments were microinjected into F2 hybrid zygotes from C57BL/6J X SJL/J parents at a concentration of 2-3 ng/µl. Injections were done by the Transgenic Animal Model Core in the University of Michigan Biomedical Core Research Facility. All procedures using mice were approved by the University of Michigan Committee on Use and Care of Animals, and all work was conducted in accord with the principles and procedures outlined in the National Institutes of Health Guidelines for the Use and Care of Experimental Animals. Four founder animals were identified for each construct and were mated to CD-1 females to acquire F1 males that could be used in breeding for all experimental time points. Timed matings were done to obtain F2 (in some cases, F3) conceptuses for S1 analysis.

DNA Analysis

DNA for polymerase chain reaction and Southern analysis was purified from tails of founders or F1 and from the heads of F2 or F3 fetal and embryonic conceptuses. Polymerase chain reaction primers consisted of 5'-AGCTGCTGCAGTCAAAGTCGAATGCAGCTG and 5'-TCCATCCATTTCTACCATTTCTTTCTCCTA and detected the boundary between the upstream epsilon  region and HS3. For determination of copy number and transgene integrity, Southern blots were probed with a 0.4-kb HindIII/BamHI fragment corresponding to the 5' end of the 1.9-kb HindIII HS3 fragment used in both constructs.

RNA Analysis

RNA was extracted from 10.5-day yolk sacs, or from fetal liver of 12.5-, 14.5-, and 16.5-day conceptuses (the morning on which the plug was detected was considered day 0.5). Tissues were dissected and immediately frozen in liquid nitrogen prior to processing. Isolation of RNA was accomplished using Trizol (Life Technologies, Inc.) according to the manufacturer's directions. RNA was quantitated spectrophotometrically and was analyzed on agarose gels to assess integrity. To quantitate mRNA levels, S1 nuclease protection was used according to published protocols (14). S1 nuclease probes for the detection of human and mouse mRNAs were kindly provided by Dr. Timothy Ley and have been described earlier (14, 15). The galago gamma  S1 probe corresponded to a 435-bp XbaI/BamHI genomic fragment labeled at the BamHI site in exon 2. The protected fragment was 204 bp. Quantitation of the signals from S1 analysis was accomplished using a PhosphorImager with ImageQuant software.


RESULTS

Two related constructs (of structure HS3-epsilon -gamma ) were introduced into transgenic mice (Fig. 1A). The degree of homology of the two gamma  fragments used is illustrated in Fig. 1B. The native galago beta -like globin cluster contains a single gamma  gene, whereas the human cluster contains two gamma  genes (5). The human Agamma (gamma 2) gene, including its 3' enhancer (13), was used in these constructs. The galago gamma  gene contains sequences similar to the human Agamma enhancer as indicated in the matrix plot of homology shown in Fig. 1B; however, the regulatory function, if any, of this region of the galago gamma  gene has never been tested.


Fig. 1. A, composition of transgenes. Restriction fragments used in the construction are indicated, and the corresponding GenBank coordinates are listed under "Experimental Procedures." B, homology between human Agamma and galago gamma  genes used in these experiments. An arrowhead marks the start of transcription of the gamma  gene. Note that sequences homologous to the human Agamma enhancer are present in galago (brackets).
[View Larger Version of this Image (16K GIF file)]

LCR sequences are necessary for the high level expression of human transgenes in the murine background (16), but single DNaseI hypersensitive sites (HS) within the LCR can also confer this property (17-20). Because it had been demonstrated that HS3 could impart high level, copy number dependent expression to a human transgene, a 1.9-kb HindIII fragment spanning this region was included in both constructs (17, 19). In addition, earlier data indicated that of all of the hypersensitive sites, HS3 may be uniquely able to drive gamma  expression in the fetal liver (20). To provide a standard against which to compare expression of the human and galago gamma  genes, the human epsilon  gene (-2000 to +1780) was also included in both constructs. Earlier studies had shown that this epsilon  fragment is expressed in the embryonic yolk sac and silenced autonomously in the fetal liver (21, 22).

Transgene copy number (Table I) and integrity (not shown) were assessed in Southern blots of tail DNA. For each construct, transgenic males from four independent lines were bred to obtain embryonic and fetal tissues. Table I summarizes the copy number corrected expression levels for epsilon  and gamma  transgenes in all eight lines examined relative to total mouse alpha  chains. S1 nuclease analysis of epsilon  and gamma  expression in a representative transgenic line carrying each type of gamma  gene is shown in Fig. 2.

Table I. Summary of transgene expression


Line Copy number Daya  gamma Expression levelb Fold gamma  down-regulationc  varepsilon Expression levelb Fold varepsilon  down-regulationc

HS3-varepsilon -galago gamma  
  2789 15 10.5  (YS) 5.3  ± 1.8 (2) 265 16.2  ± 10.8 (2) 116
12.5  (YS) 0.5  ± 0.2 (4) 3.2  ± 0.99 (5)
14.5  (FL) 0.07  ± 0.05 (7) 0.21  ± 0.15 (8)
16.5  (FL) 0.02  ± 0.01 (2) 0.14  ± 0.06 (4)
  2670 1 10.5  (YS) 24.5  ± 9.2 (4) 3.3 156  ± 31 (4) 24
12.5  (YS) 9.7  ± 3.3 (5) 72  ± 14 (5)
14.5  (FL) 8.0  ± 2.3 (4) 5.6  ± 2.9 (4)
16.5  (FL) 7.4  ± 4.2 (4) 6.5  ± 0.4 (3)
  2727 35 10.5  (YS) 0.14  ± 0.11 (4) 4.7 5.0  ± 2.8 (4) 31.2
12.5  (YS) 0.04  ± 0.03 (5) 1.9  ± 0.7 (5)
14.5  (FL) 0.01  ± 0.007 (4) 0.15  ± 0.04 (4)
16.5  (FL) 0.03  ± 0.009 (7) 0.16  ± 0.03 (7)
  2674 5 10.5  (YS) 15.8  ± 6.8 (6) 198 44.6  ± 10.2 (6) 1487
12.5  (YS) 5.1  ± 2.4 (6) 20.6  ± 17.2 (6)
14.5  (FL) 0.24  ± 0.1 (4) 0.2  ± 0.11 (4)
16.5  (FL) 0.08  ± 0.04 (8) 0.03  ± 0.04 (8)
HS3-varepsilon -human gamma  
  2564 10 10.5  (YS) 25  ± 4 (3) 1.8 21  ± 3 (3) 61.8
12.5  (YS) 55  ± 22 (3) 9.5  ± 2.5 (3)
14.5  (FL) 126  ± 59 (4) 0.72  ± 0.12 (3)
16.5  (FL) 69  ± 44 (2) 0.34  ± 0.04 (2)
  2649 25 10.5  (YS) 6.8  ± 2.5 (2) 2.2 3.2  ± 1.2 (2) 15.3
12.5  (YS) 29  ± 13 (3) 5.8  ± 0.6 (3)
14.5  (FL) 17  ± 8 (3) 0.33  ± 0.14 (3)
16.5  (FL) 7.8  ± 4.3 (3) 0.15  ± 0.05 (3)
  155 7 10.5  (YS) 9.2  ± 1.3 (3) 1.6 5.0  ± 3.0 (3) 16.7
12.5  (YS) 11  ± 4 (3) 5.6  ± 2.3 (3)
14.5  (FL) 12  ± 9 (3) 0.25  ± 0.06 (3)
16.5  (FL) 7.6  ± 1.6 (3) 0.3  ± 0.17 (3)
  150 2 10.5  (YS) 41  ± 20 (2) 1.1 28  ± 9 (2) 23.3
12.5  (YS) 19  ± 2 (3) 9.4  ± 1.8 (3)
14.5  (FL) 138  ± 36 (3) 1.6  ± 0.3 (2)
16.5  (FL) 117  ± 41 (3) 1.2  ± 0.8 (3)

a Days after observation of plug. YS, yolk sac; FL, fetal liver.
b Copy number corrected expression (e.g., [gamma /copy number]/([mouse alpha /4] + [mouse zeta /2])).
c Fold down-regulation as (highest expression level)/(expression level at 16 days).


Fig. 2. Developmental pattern of transgene expression as detected by S1 nuclease protection assays of individual offspring from transgenic lines carrying HS3-epsilon -humgamma and HS3-epsilon -galgamma constructs. A, HS3-epsilon -humgamma lines. The protected fragments corresponding to human epsilon  (hepsilon ), mouse alpha  (malpha ), and human gamma  (humgamma ) are shown at four time points in development (10.5-day yolk sac, 12.5-day yolk sac, 14.5-day fetal liver, and 16.5-day fetal liver). Multiple lanes represent independent transgenic embryos or fetuses from the same litter; all lanes are offspring derived from founder 2564. B, HS3-epsilon -galgamma lines. Protected fragments arising from human epsilon  (hepsilon ), mouse alpha  (malpha ), and galago gamma  (galgamma ) transcripts are labeled. The unlabeled band between the malpha and galgamma bands arises from protection of a mouse beta h1 probe. Time points and tissue sources are as described for A; all were taken from line 2674. All assays utilized 500 ng of RNA. That the assays were performed under conditions of probe excess was verified by re-examination of several samples with 1.5 µg of RNA (not shown).
[View Larger Version of this Image (70K GIF file)]

All eight transgenic lines expressed both the epsilon  and gamma  transgenes. However, line to line variation in transgene expression level was observed, most likely due to position effects. Thus, expression was not copy number-dependent despite the fact that both constructs contained the region of HS3 recently shown to possess dominant chromatin opening function (23). Significant position effects with HS3Agamma transgenes (but missing the Agamma enhancer) have also been observed by others (24). Interestingly, all four HS3-epsilon -galgamma lines and three of four HS3-epsilon -humgamma lines exhibit an inverse relationship between copy number and expression (Table I). This pattern has been observed previously with HS2-containing constructs (25), but the significance of this phenomenon is presently unclear.

Although expression levels varied, patterns of transgene expression during development were highly reproducible for each gene as illustrated in Fig. 3 where expression at each time point is plotted relative to the 10.5-day expression level (which is taken as 100%). In both HS3-epsilon -humgamma and HS3-epsilon -galgamma lines, the human epsilon  gene was expressed at high levels in the embryonic yolk sac (day 10.5 and 12.5) and was significantly repressed in 14.5 and 16.5 day fetal livers (Figs. 2 and 3). The yolk sac portion (10.5 and 12.5 days) of the epsilon  expression curves in HS3-epsilon -humgamma lines were somewhat more variable than those seen in HS3-epsilon -galgamma lines. However, the well known variability in the timing of development of conceptuses even within the same litter makes it difficult to determine if these differences are significant. Nevertheless, the fetal portion (14.5 and 16.5 days) of the epsilon  expression curves was identical in mice carrying both constructs; the human epsilon  gene was silenced in fetal life.


Fig. 3. Comparison of expression patterns for human epsilon , galago gamma , and human gamma  genes. Data in Table I are plotted using the 10.5 day embryonic level as 100% and normalizing all other points to this level. Dotted lines indicate gamma  expression; solid lines indicate epsilon  expression.
[View Larger Version of this Image (22K GIF file)]

In contrast, the two gamma  genes exhibited distinctly different expression patterns in the fetal liver (Figs. 2 and 3). The galago gamma  gene was expressed at highest levels in embryonic life and silenced along with the human epsilon  gene by 14.5 days, mimicking the embryonic pattern characteristic of the galago (5). Interestingly, the developmental expression curves for human epsilon  and galago gamma  in each line were nearly superimposed, suggesting that the two genes were coordinately silenced. In contrast, the human gamma  gene was not coordinately silenced with epsilon ; rather, expression peaked in 14.5 day fetal livers and declined at 16.5 days. Although expression curves were somewhat variable in shape, considerable gamma  expression was still observed at 16.5 days, a pattern distinctly different than that seen for the galago gamma  gene.

Examination of 10.5-day expression levels for all transgenes (Table I) reveals that in HS3-epsilon -humgamma lines, gamma  gene expression was greater than epsilon  gene expression (average 1.4-fold). This pattern (epsilon  < gamma ) has been seen by others when larger constructs containing the human epsilon  and gamma  genes were studied in the mouse (26, 27). In contrast, in HS3-epsilon -galgamma lines, human epsilon  expression was greater than galago gamma  expression (3.3-, 6.4-, 35.7-, and 2.8-fold for the four lines).


DISCUSSION

These data indicate that the characteristic embryonic expression pattern of the galago gamma  gene can be recapitulated in the transgenic mouse. It has also been demonstrated that globin genes from the chicken (28) and frog (29) are expressed in the mouse background in temporal patterns similar to those expected on the basis of in vivo patterns. Together, these studies attest to the broad evolutionary conservation of cis and trans regulators of globin gene expression.

The work presented here demonstrates that human and galago gamma  transgenes exhibit different developmental expression patterns when linked to the same portion of the LCR and when placed in the same microenvironment (mouse fetal liver). The divergent expression patterns of the two genes must therefore be due to differences in DNA sequence (cis elements) within the 4.0-kb fragment that contains the gamma  gene. Thus, fetal recruitment of the simian gamma  gene was (at least in part, see below) a cis-mediated event. Moreover, this result confirms that cis signals for stage-specific globin gene expression must reside near the genes, not within the LCR. Earlier studies of human transgene expression in the absence of LCR sequences also support this conclusion (30).

The data also eliminate distance from the LCR as a determinant per se of the differences in stage-specific gene expression of these two genes (31, 32). The physical distance between the gamma  gene(s) and the LCR in the intact beta -like globin loci of human and galago differ significantly; the single galago gamma  gene is 13.5 kb from the 3' end of the LCR (the HS1 core), whereas the human Ggamma and Agamma genes are 21 and 26 kb away, respectively. In the constructs studied here, both gamma  genes were equidistant from HS3; however, their characteristic expression patterns were preserved. It is nevertheless possible that the increased distance of the duplicated human gamma  gene from the LCR may have played a permissive role in the initial evolution of a new fetal expression pattern (6, 11).

The conclusion that a fetal liver trans environment that is permissive for gamma  expression had already evolved prior to the mammalian radiation is supported by data presented here and elsewhere (24, 26, 27, 33). This does not imply that the mouse fetal liver environment is identical to that of the human; differences may exist in the relative balance of trans factors that would result in some distinct patterns of regulation in each species. Indeed, when the human gamma  gene is placed in the context of the entire beta -like globin locus, it seems to be silenced at an earlier developmental time in the mouse fetal liver than in the human fetal liver (26, 27). Regardless of these differences, the data presented here indicate clearly that cis differences exist between galago and human gamma  genes that result in the generation of distinct patterns of expression in the fetal mouse liver; the galago gamma  gene is silenced, whereas human gamma  gene expression peaks in this stage.

Interestingly, in several independent lines carrying the HS3-epsilon -galgamma construct, the kinetics of galago gamma  and human epsilon  gene silencing after embryonic life were nearly identical. Such coordinate regulation could be a consequence of lineage restriction. That is, both genes may be expressed at high levels only in yolk sac derived "primitive" erythrocytes and not in fetal liver-derived "definitive" erythrocytes. Whether there are actually two different stem cell lineages that contribute progeny to primitive and definitive lineages is still a matter of some debate, but recent identification of an intraembryonic source of long term repopulating hematopoietic cells suggests that this is likely (reviewed in Ref. 34). Coordinate regulation of human epsilon  and galago gamma  genes could be achieved by the presence of silencers that act on both genes in definitive cells or by the absence of primitive activators in definitive cells. Alternatively, lineage specific changes in chromatin structure may explain the coordinate silencing of these two genes. The human gamma  gene but not the human epsilon  or galago gamma  genes may contain elements that allow it to be expressed in the progressively heterochromatic environment of the definitive cell.

In order for the simian gamma  gene to complete the transition from an exclusively embryonically expressed gene (the galago gamma  pattern) to a primarily fetally expressed gene (the human gamma  pattern), a second anthropoid-specific change is required: reduction of embryonic expression levels. This could have been accomplished by cis alterations that created binding site(s) for anthropoid-specific embryonic repressor(s) or by trans changes (loss of an embryonic activator of the gamma  gene specifically in anthropoid primates). Both scenarios imply that the trans environment of the mouse yolk sac must differ from that of the human and other anthropoid primates. Because gamma  globin gene expression has only been studied in relatively few anthropoid primates, it is possible that further analysis will reveal a species in which the gamma  gene is expressed at high levels in both embryonic and fetal life.

The constructs described here should facilitate the identification of the specific cis sequence change(s) that mediated fetal gamma  expression, information that will likely reveal the molecular mechanisms responsible for acquisition of this new temporal expression domain. Several possible mechanisms exist, and a few candidate cis elements have already been identified. First, nucleotide changes could have resulted in the loss of fetal-specific repressor binding site(s) in the ancestral simian gamma  gene; a region near the proximal CCAAT box shows anthropoid-specific base changes that reduce the binding of a complex of putative fetal repressor proteins (35). Second, base changes could have generated simian-specific activator motif(s); anthropoid-specific changes in the -1086 region alter a YY1 binding site that appears to be important for the activation of gamma  in the fetal stage.2 Third, the gain of a binding site for a fetal stage selector protein (SSP; Ref. 36) may have given the gamma  gene a competitive edge over the beta  gene in fetal life. In the -50 region of the human gamma  promoter, several anthropoid-specific nucleotides comprise a binding site for SSP; the SSP site is absent in the galago gamma  gene (36). Finally, fetal gamma  expression could have arisen via acquisition of a new interaction between the gamma  promoter and the LCR that is stable in the fetal stage. In this regard, it is of interest that in HS3-epsilon -galgamma lines, epsilon >gamma and the two genes are coordinately silenced, but in HS3-epsilon -humgamma lines, gamma  > epsilon  and silencing is not coordinate. Establishment of a strong LCR contact that is stable in fetal life would not only accomplish the fetal recruitment of gamma  but could conceivably force a delay in the expression of beta  via competitive mechanisms. Indeed, it has been demonstrated that the galago beta  gene is activated in early fetal life, whereas human beta  gene activation occurs at birth (5). Identification of the exact cis sequences that mediated the different expression patterns of the gamma  genes observed in this study is likely to further our understanding of the molecular mechanisms that control the evolution of novel stage-specific expression domains as well as the regulation of hemoglobin switching.


FOOTNOTES

*   This work was supported by Public Health Service Grants NIH-HL48802 (to D. L. G.) and NIH-HL 33940 (to M. G.). Computer facilities were provided by the General Clinical Research Center (University of Michigan) supported by a Grant NIH-M01RR00042 from the National Center for Research Resources.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
par    To whom correspondence should be addressed: University of Michigan, Dept. of Anatomy and Cell Biology, 5793 Medical Science II, Ann Arbor, MI 48109-0616. Tel.: 313-647-0172; Fax: 313-763-1166; E-mail: dgumucio{at}umich.edu.
1   The abbreviations used are: MYA, million years ago; LCR, locus control region; bp, base pair(s); kb, kilobase(s); HS, hypersensitive site; SSP, stage selector protein.
2   W. Zhu, C. TomHon, N. Richards, D. Fairfield, and D. L. Gumucio, submitted for publication.

ACKNOWLEDGEMENTS

We thank Drs. Sally Camper, Linda Samuelson, and Kevin McDonagh for comments and suggestions, J. Lloyd for the HS3 fragment, T. Ley for clones used as S1 probes, and the University of Michigan Transgenic Mouse Core for the production of transgenic lines.


REFERENCES

  1. Koop, B. F., and Goodman, M. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 3893-3897 [Abstract]
  2. Cooper, S. J. B., and Hope, R. M. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 11777-11781 [Abstract]
  3. Goodman, M., Koop, B. F., Czelusniak, J., Weiss, M. L., and Slightom, J. L. (1984) J. Mol. Biol. 108, 803-823
  4. Hardison, R. C. (1984) Mol. Biol. Evol. 1, 390-410 [Abstract]
  5. Tagle, D. A., Koop, B. F., Goodman, M., Slightom, J. L., Hess, D. L., and Jones, R. T. (1988) J. Mol. Biol. 203, 439-455 [Medline] [Order article via Infotrieve]
  6. Goodman, M., Slightom, J. L., and Gumucio, D. L. (1996) in Gene Families: Structure, Function, Genetics and Evolution (Holmes, R. S., and Lim, H. A., eds), pp. 43-52, World Scientific Publishing Co., River Edge, New Jersey
  7. Hill, A., Hardies, S. C., Phillips, S. J., Davis, M. G., Hutchison, C. A., III, and Edgell, M. H. (1984) J. Biol. Chem. 259, 3739-3747 [Abstract/Free Full Text]
  8. Goodman, M. (1981) Prog. Biophys. Mol. Biol. 37, 105-164
  9. Czelusniak, J., Goodman, M., Hewett-Emmett, D., Weiss, M. L., Venta, P. J., and Tashian, R. E. (1982) Nature 298, 297-300 [Medline] [Order article via Infotrieve]
  10. Goodman, M., Czelusniak, J., Koop, B. F., Tagle, D. A., and Slightom, J. L. (1987) Cold Spring Harbor Symp. Quant. Biol. 522, 875-890
  11. Fitch, D. H. A., Bailey, W. J., Tagle, D. A., Goodman, M., Sieu, L., and Slightom, J. L. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 7396-7400 [Abstract]
  12. Hayasaka, K., Skinner, C. G., Goodman, M., and Slightom, J. L. (1993) Genomics 18, 20-28 [CrossRef][Medline] [Order article via Infotrieve]
  13. Bodine, D. M., and Ley, T. J. (1987) EMBO J. 6, 2997-3004 [Abstract]
  14. Ulrich, M. J., and Ley, T. J. (1990) Blood 75, 990-999 [Abstract]
  15. Ley, T. J., Maloney, K. A., Gordon, J. I., and Schwartz, A. L. (1989) J. Clin. Invest. 83, 1032-1036 [Medline] [Order article via Infotrieve]
  16. Grosveld, F., van Assendelft, G. B., Greaves, D. R., and Kollias, G. (1987) Cell 51, 975-985 [Medline] [Order article via Infotrieve]
  17. Fraser, P., Hurst, J., Collis, P., and Grosveld, F. (1990) Nucleic Acids Res. 18, 3503-3507 [Abstract]
  18. Ryan, T. M., Behringer, R. R., Martin, N. C., Townes, T. M., Palmiter, R. D., and Brinster, R. L. (1989) Genes & Dev. 3, 314-323 [Abstract]
  19. Philipsen, S., Talbot, D., Fraser, P., and Grosveld, F. (1990) EMBO J. 9, 2159-2167 [Abstract]
  20. Fraser, P., Pruzina, S., Antoniou, M., and Grosveld, F. (1993) Genes & Dev. 7, 106-113 [Abstract]
  21. Shih, D. M., Wall, R. J., and Shapiro, S. T. (1990) Nucleic Acids Res. 18, 5465-5472 [Abstract]
  22. Raich, N., Enver, T., Nakamoto, B., Josephson, B., Papayannopoulou, T., and Stamatoyannopoulos, G. (1990) Science 250, 1147-1149 [Medline] [Order article via Infotrieve]
  23. Ellis, J., Kian, C. T.-U., Harper, A., Michalovich, D., Yannoutsos, N., Philipsen, S., and Grosveld, F. (1996) EMBO J. 15, 562-568 [Abstract]
  24. Li, Q., and Stamatoyannopoulos, G. (1994) Mol. Cell. Biol. 14, 6087-6096 [Abstract]
  25. Morley, B. J., Abbott, C. A., Sharpe, J. S., Lida, J., Chan-Thomas, P. S., and Wood, W. G. (1992) Mol. Cell. Biol. 12, 2057-2066 [Abstract]
  26. Strouboulis, J., Dillon, N., and Grosveld, F. (1992) Genes & Dev. 6, 1857-1864 [Abstract]
  27. Peterson, K. R., Clegg, C. H., Huxley, C., Josephson, B. M., Haugen, H. S., Furukawa, T., and Stamatoyannopoulos, G. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 7593-7597 [Abstract/Free Full Text]
  28. Mason, M. M., Kee, E., Westphal, H., and Reitman, M. (1995) Mol. Cell. Biol. 15, 407-414 [Abstract]
  29. Dillon, N., Kollias, G., Grosveld, F., and Williams, J. G. (1991) Nucleic Acids Res. 19, 6227-6230 [Abstract]
  30. Starck, J., Sarkar, R., Romana, M., Bhargava, A., Scarpa, A. L., Tanaka, M., Chamberlain, J. W., Weissman, S. M., and Forget, B. G. (1994) Blood 84, 1656-1665 [Abstract/Free Full Text]
  31. Peterson, K. R., and Stamatoyannopoulos, G. (1993) Mol. Cell. Biol. 13, 4836-4843 [Abstract]
  32. Hanscomb, O., Whyatt, D., Fraser, P., Yannoutsos, N., Greaves, D., Dillon, N., and Grosveld, F. (1991) Genes & Dev. 5, 1387-1394 [Abstract]
  33. Dillon, N., and Grosveld, F. (1991) Nature 350, 252-255 [CrossRef][Medline] [Order article via Infotrieve]
  34. Dzierzak, E., and Medvinsky, A. (1995) Trends Genet. 11, 359-365 [CrossRef][Medline] [Order article via Infotrieve]
  35. Gumucio, D. L., Shelton, D. A., Blanchard-McQuate, K., Gray, T., Tarle, S., Heilstedt-Williamson, H., Slightom, J. L., Collins, F., and Goodman, M. (1994) J. Biol. Chem. 269, 15371-15380 [Abstract/Free Full Text]
  36. Jane, S. M., Ney, P. A., Vanin, E. F., Gumucio, D. L., and Nienhuis, A. W. (1992) EMBO J. 11, 2961-2969 [Abstract]

©1997 by The American Society for Biochemistry and Molecular Biology, Inc.