We are here to honor a group of pioneers in mammalian genetics for their achievements in developing the inbred mouse as a genetic tool. in doing so we honor them not only for the significance their achievement has for today, but also for the even greater promise it has for the future. That promise comes from the application of the inbred mouse and its genetics to new questions, questions whose answers could only be dreamed about in the early days of mammalian genetics. Perhaps the most graphic tribute we can give is to describe these applications. One of them is the study of gene regulation that grew out of a marriage between mammalian genetics and biochemistry. It is interesting that both fields had their origins at very nearly the same time. The study of intermediary metabolism began with the discovery of the first sugar phosphate in 1905, almost simultaneous with the beginnings of mouse genetics. However, it was not until 1941 that Dr. Strong first reported an enzyme difference between mouse strains ( 1), in what is to my knowledge the first report in any organism of an enzyme variant, and it was in 1952 that Dr. Law carried out the first genetic analysis of an enzyme difference ( 2), describing the inheritance pattern of the low glucuronidase phenotype that has occupied so much attention over subsequent years.
My task is to illustrate the types of genetic variation that influence enzyme activity and the experimental approaches, both genetic and biochemical, used to analyze them. Because the subject is too large to encompass in one brief talk, the illustrations are necessarily selected. I shall put some emphasis on β-glucuronidase because it is the most extensively studied system and also the one with which I am most familiar. Various aspects of the genetics ( 3), biochemistry and intracellular localization ( 4), and hormonal induction ( 5) of this enzyme have been reviewed.
Over the years it has proved both conceptually and operationally useful to divide enzyme variants into four groups. The composition of these groups has varied slightly from time to time as additional insights have been gained. Table 1 lists these categories and the genes that are known to fall into one or another category with the exception that I have not listed the many structural mutants known. There are simply too many of these and a separate catalogue of them is available ( 6). Of course, to some measure the assignment of a locus to one or another category depends on our frame of reference. For example, the Eg gene is almost certainly the structural gene for the protein egasyn, but is listed here among the processing genes for its role in the intracellular localization of β-glucuronidase. Similarly, the Tfm locus, that is probably the structural gene for the androgen receptor protein, is listed here among regulatory genes for its role in regulating hormonal induction of other proteins.
One group of interesting, but not easily categorized mutants is the series of radiation-induced deletions overlapping the albino locus. Their biochemical phenotypes have been extensively studied by Gluecksohn-Waelsch and co-workers ( 7, 8, 9, 10). Using complementation testing in heterozygotes, it has become possible to define a number of subregions with distinct biochemical functions within one short distance.
Dr. Hutton has just discussed structural genes in considerable detail. I shall only point out one additional aspect in relation to determination of enzyme activity. A great deal of intracellular processing of polypeptide chains occurs post-translationally, and this processing is quite significant in the eventual realization of enzyme function (see below). Appropriate processing requires that each polypeptide sequence be recognized correctly, so amino acid substitutions within the recognition region may alter processing and eventually enzyme function. One such case has been described. A structural mutation in β-glucuronidase influences its partition between two intracellular compartments.
β-glucuronidase is present in lysosomes in virtually all cells except erythrocytes, and in some tissues is also present in endoplasmic reticulum ( 11, 12). In liver, for example, nearly half of the enzyme is membrane bound. Genetic, biochemical, and immunological evidence indicate that the enzyme from both sites is derived from the same structural gene ( 11, 13, 14, 15), located near the distal end of chromosome 5 ( 11, 16, 17, 18, 19). The product of this structural gene is an enzyme tetramer of four identical subunits that is glycosylated and has been purified to homogeneity ( 21). Three alleles of the structural gene have been described in the literature, Gusb, the standard form of the enzyme, Gusa, an electrophoretic variant with more rapid mobility, and Gush, a variant with decreased thermostability. Several additional alleles are known, including one with increased thermostability and another with a new electrophoretic mobility ( 20). With respect to enzyme localization it has turned out that not all allelic forms of the enzyme respond equally to the intracellular mechanisms involved, and that the GUS-H allozyme is not distributed between the two sites in the same manner that other enzyme forms are ( 11, 22), presumably because of a change in its recognition properties.
Among the second category of genes in earlier versions of Table 1 ( 22, 23) only loci affecting intracellular localization were included; these were considered architectural. Subsequent experience suggests that the second category should be enlarged to include a variety of mutations affecting the post-translational life of an enzyme molecule.
Modification. The final completion of enzyme chains by post-translational modification includes a variety of processes including proteolytic cleavages and addition of a wide variety of conjugant groups. Several mutants have been reported that are altered in enzyme conjugation.
The acid α-mannosidase present in lysosomes carries sialic acid in liver, but not in kidney. All mouse strains appear to have enzyme molecules with the same electrophoretic mobility in kidney, but vary in extent of sialylation and hence electrophoretic mobility of the liver enzyme; at least two loci are involved ( 24, 25), the Map-1 gene located on chromosome 17 near the H-2 complex and the Map-2 gene on chromosome 5 near Gus. The Map-1 locus may be identical with the Apl locus described by Lalley and Shows ( 26) which affects the electrophoretic mobility of acid phosphatase in liver but not in kidney. These genes probably determine the presence of specific sialyl transferases. Their existence makes the interesting point that a family of transferases probably exists, each with a spectrum of enzymes it is capable of sialylating, since other acid hydrolases that are sialylated in liver but not in kidney are not affected by variation at these loci ( 27, 28, 29).
Turnover. Rates of enzyme synthesis and degradation have an equal influence on the final concentration of enzyme activity in a tissue ( 30). However, it is not clear that there is much specificity in the system used for intracellular protein turnover, or that variations in protein turnover are used as a regulatory mechanism for individual proteins. Although the turnover of lactate dehydrogenase varies considerably from one tissue to another, the ratio of lactate dehydrogenase turnover to the turnover of average cellular proteins remains nearly the same from one tissue to another. Thus, modulations in relative concentrations of the enzyme must be achieved by altering its synthesis relative to that of other proteins ( 31). It is not surprising, then, that only one variant has so far been described with a specific alteration in the intracellular degradation of a protein. This is the Ce locus determining the rate of catalase degradation in liver ( 32, 33). Several substrains of the C57 family have twice the catalase activity of other strains as a result of decreased enzyme turnover in liver but not in kidney. Fast versus slow turnover segregates as a single Mendelian factor with fast turnover dominant. The locus involved is not linked to the structural gene.
Architectural. Approximately half of all cellular protein exists incorporated into one cellular organelle or another, but relatively little is known as to how this is achieved beyond the recent discovery that N-terminal peptide sequences act as signals deciding whether nascent chains attach to membranes ( 34) and the genetic evidence for a special class of anchor proteins involved in localization. This evidence comes from the only mutation affecting intracellular localization that has been described and involves the Eg locus and β-glucuronidase localization ( 14). This lack of mutants may reflect the difficulty of recognizing mutants with localization defects since failure to successfully locate an enzyme may not cause an obviously different phenotype from failure to synthesize the protein in the first place. It was possible to detect the Eg mutant because β-glucuronidase is unusual in being present in both lysosomes and endoplasmic reticulum. The mutant was detected as a loss of enzyme from the endoplasmic reticulum site without loss of enzyme from the lysosomal site. The properties of this mutant and the mechanisms of intracellular localization of glucuronidase have been reviewed recently ( 4).
Briefly, the polypeptide product of the single glucuronidase structural gene, Gus, is processed and assembled into two distinct tetrameric forms of the enzyme, L and X, that differ slightly in both charge and molecular weight ( 15, 35, 36). The L form is almost entirely located in lysosomes where it is the only enzyme form present. The X form is entirely located in endoplasmic reticulum, where it occurs complexed with one to four molecules of the protein egasyn ( 15, 36). Formation of these complexes, or M forms, is apparently required to anchor or stabilize the binding of X to the membrane ( 37). Mutation in the Eg gene results in loss of egasyn ( 38, 39) and the absence of enzyme bound to membranes ( 14, 40). Eg is located on chromosome 8 unlinked to the glucuronidase structural gene ( 14, 41). Heterozygotes have reduced levels of egasyn but normal binding of enzyme to membrane ( 39). Only tissues containing egasyn possess membrane bound glucuronidase ( 4, 42).
Organellar Function. Once a protein has become integrated into a cellular organelle its fate, at least in part, is tied to that of the organelle itself. A case in point is the series of mutants described by Swank and co-workers ( 43) that appear to be deficient in the transport of lysosomes across cell membranes. The initial observation was that the beige mutant, which possesses enlarged lysosomes in many cells and is an analog of Chediak-Higashi syndrome in the human, accumulates excessive amounts of β-glucuronidase during induction of this enzyme in kidney proximal tubule epithelial cells ( 44, 45). Normally these cells excrete most of their induced enzyme by a process that appears to involve extrusion of entire lysosomal contents into the lumen of the collecting tubule. The beige mutant is deficient in carrying out this process and consequently accumulates large amounts of enzyme. Following the observation by Meisler ( 46) that another pigmentation mutant, light ears, shares a similar defect, four additional pigmentation mutants were found defective in lysosome transport ( 43). Lysosomes and melanosomes apparently share common mechanisms of transport across membranes. The various mutations are unlinked and recessive, and presumably affect different functional steps in the process of transport.
Regulatory genes are distinguished by their effect on protein synthesis rather than merely altering apparent enzyme activity. Activity changes can result from a variety of causes that do not involve true regulation, including alterations in processing and structure. At the present time it is useful to distinguish between regulatory sites controlling systemic levels of enzyme, those determining the response of an enzyme to an effector molecule such as a hormone, and those affecting the receptor molecules involved in effector responses.
Systemic. There are now two examples of genetic variants in which enzyme activity is altered in a more or less constant manner among all tissues and at different stages of development. The first discovered was the Lv locus determining levels of aminolevulate dehydratase in several tissues during both fetal and adult stages of development ( 47). High strains have several times the activity of low strains and heterozygotes are intermediate. A third allele with intermediate levels has been reported ( 48). The locus is linked to the enzyme structural gene ( 49). Enzyme purified from both high and low strains has identical kinetic and physical properties and the same catalytic activity per molecule ( 50, 51). Labeling studies have shown that the kinetics of enzyme turnover are the same in high and low strains and that the difference lies in the rate of enzyme synthesis.
A systemic regulatory locus for β-galactosidase has also been found that in many respects resembles the Lv locus. It acts uniformly in different tissues and stages of development, is closely linked to the structural gene, and controls enzyme activity by regulating enzyme synthesis. The structural gene for murine β-galactosidase is defined by an electrophoretic polymorphism, Bge, and is located on chromosome 9 ( 27). Although multiple forms of β-galactosidase are present in tissues, these are all derived from this single structural gene, and represent charge differences arising from differential sialylation of the enzyme among tissues as well as several molecular weight forms resulting from pH dependent aggregation of the enzyme ( 27). The systemic regulator, Bgs, is very closely linked to Bge, and no recombination was observed between the two ( 27). Strains carrying the Bgsh allele have approximately twice the enzyme level of Bgsd strains in a variety of tissues throughout development ( 52, 53). Heterozygotes show additive inheritance, possessing intermediate enzyme levels. Antibody titration shows that high enzyme activity reflects and increase in the number of enzyme molecules present ( 52) and recent labeling experiments show this results from increased enzyme synthesis ( 54).
The difference in activity seen between Bgsd and Bgsh strains has been used as a histochemical marker for the recognition of genetically marked cells in chimeric mice ( 55).
Induction. Mutants with altered response to enzyme induction have received considerable attention for the insights they offer into mechanisms of regulation of gene transcription and the control of physiological responses in tissues. Three systems have been examined so far, all involving induction by androgens. These include glucuronidase induction in epithelial proximal tubule cells of kidney, induction of rennin activity in submandular gland, and induction of the major urinary proteins (Mups) in liver.
A massive increase in the glucuronidase content of kidney proximal tubule epithelial cells occurs following administration of androgens in the mouse ( 19, 56, 57). Several other enzymes, notably D-amino acid oxidase and alcohol dehydrogenase, are also induced, but to lesser extents. There is a very small induction of other lysosomal hydrolases. Much of the induced glucuronidase is excreted into urine. Electron micrographs ( 3) show the accumulation of membranous material inside lysosomes and large amounts of similar material appearing in the proximal tubule lumen.
Inbred strains fall into two major classes of high and low inducibility ( 19). Additional rare phenotypes may exist ( 20). The system has been characterized in some detail using A/J and C57BL/6J as the prototype high and low strains ( 19). Among the androgen-inducible proteins only glucuronidase is affected, and the difference in inducibility is independent of the specific hormone used and the dosage.
Examination of recombinant inbred lines between BALB/c and C57BL/6 and crosses between A/J and C57BL/6 showed that inducibility is controlled by a single locus, Gur, closely linked to the structural gene ( 19, 58). Despite differences in reported properties, the O locus described by Ohno and coworkers ( 59) is probably identical with Gur. Originally several ostensible recombinants were observed between the new regulatory site and the structural gene ( 19), but true recombinants were not found in a more extensive analysis of this cross using newer techniques allowing the survival of test animals and subsequent breeding ( 5). Ostensible recombinants did arise at very low frequency but proved to be errors of diagnosis.
Pulse labeling studies have shown that Gur locus control is a cis acting regulation of enzyme synthesis ( 19). Heterozygotes carrying Gura/ Gurb have intermediate levels of enzyme because one chromosome is producing enzyme rapidly and the other slowly.
The analysis of recombinant inbred lines showed the presence of genes that affect the final level of induced enzyme activity, but not the early kinetics of induction ( 58). Labeling studies showed that unlike Gur these genes do not act at the level of enzyme synthesis and a very likely affect enzyme excretion ( 60).
Molecular studies now give some indication as to the nature of Gur gene function. A very sensitive enzymatic assay for glucuronidase mRNA has been developed based on the formation of catalytically active mouse enzyme in Xenopus oocytes following the injection of mouse mRNA ( 61). Measurements of mRNA levels were made in fully induced A/J and C57BL/6 mice as well as in a congenic strain carrying the Gura allele of strain A backcrossed onto the C57BL/6 background. They showed that induction is an accumulation of mRNA activity and that the Gur locus determines the amount of mRNA activity present ( 62). Thus, the Gur locus is a regulatory site controlling mRNA activity. Detailed kinetic studies of the changes in rates of enzyme synthesis and levels of mRNA activity during induction and deinduction have been carried out ( 63). They suggest that Gur controls mRNA activity by determining the hormonal response of a rate limiting factor in the synthesis of messenger activity and that this factor turns over very slowly. The most obvious candidate for such a factor is the activation of chromatin, but this is still a hypothesis.
Several additional genes are known to affect glucuronidase induction. One is the Tfm mutant that is deficient in androgen binding protein ( 64, 65, 66) and hence loses induction ( 67). Several others act through their effects on the pituitary. Hypophysectomized animals are only inducible to about 5% of the level of intact animals ( 68), and hormone replacement experiments suggest that the missing factor is growth hormone ( 69). In accord with this, induction is lost in the dwarf mutant that lacks all pituitary function, as well as in the little mutant that is relatively specific deficiency in growth hormone ( 69). These mutations all affect enzyme synthesis.
Because enzyme excretion is the major mechanism for removal of induced enzyme, genetic factors affecting excretion influence enzyme accumulation. This is apparent in the segregation among recombinant inbred strains of genes unlinked to the structural gene ( 58), and the elevated enzyme accumulation seen in beige and other pigmentation mutants defective in lysosome excretion (see Processing above).
Rennin induction in submaxillary gland is also controlled by a single locus with additive inheritance that acts at the level of enzyme synthesis ( 70). There are multiple rennin isozymes, and both basal and induced activities are genetically affected, making it uncertain where in the induction process the gene acts.
The major urinary proteins of the mouse (Mups) are a family of three closely related proteins that are synthesized in the liver and excreted in the urine. Their interest lies in the very large synthesis achieved after androgen induction; depending upon the genetic background, this is in the range of 10-30 mg of mup protein per day, and represents the highest percentage of any single protein observed in the liver. Originally, Hudson et al. ( 71) described a locus, Mup-a, that was thought to represent a structural polymorphism in which mup-1 and mup-2 were allelic forms derived from the same structural gene. More recently Szoka and Paigen ( 72) have found that all mouse strains synthesize all three mups, and that the Mup-a locus is, in fact, regulatory. Analysis of recombinant inbred lines between BALB/c and C57BL/6, which carry the Mup-a1 and Mup-a2 alleles, respectively, has shown that the Mup-a locus determines the relative proportions of the three Mups synthesized after induction, but that other loci are involved in other aspects of the phenotype, including determination of the relative proportions before induction, the kinetics of induction, and the total level of Mup production reached at the final induced state.
Receptors. The testicular feminization mutant, Tfm, is deficient in androgen receptor protein ( 64, 65, 66) and has lost all androgen-inducible functions in the mouse. Its existence raises the question as to why, if the same receptor protein functions in all tissues, a different set of proteins is induced in each tissue. An additional specificity factor must exist.
The other receptor mutant known involves the receptor protein for planar hydrocarbons that functions in the induction of the aryl hydrocarbon hydroxylase system and some other proteins ( 73, 74). Segregation at the Ah locus determines the binding affinity of this receptor ( 75) and has important effects on the relative susceptibility of various sites to hydrocarbon induction of tumors ( 76).
Genetic factors controlling development have fascinated biologists for many years. Historically, the primary emphasis has been on morphogenetic mutants. Now, with the advent of biochemical genetics it is possible to ask questions about the genetic systems determining developmental programs for the products of individual structural genes. This approach allows us to focus on questions of programming itself and thereby avoid the confusion attending phenotypic changes that may turn out to be metabolic consequences of altered protein function. Taking this approach does not assume any particular model for genetic regulation of differentiation; rather, we appreciate that the nature of our findings will eventually help us choose between models. In this case, the mouse has led the work in other organisms. The mouse systems most extensively examined have been those controlling H-2, α-galactosidase, β-galactosidase, and β-glucuronidase. The properties of these systems and the general concepts of temporal genes have been reviewed ( 77).
The first temporal gene element identified in mice proved to be in close proximity to the glucuronidase structural gene. The developmental program for β-glucuronidase has been defined for a number of mouse tissues ( 78), and is coordinate with that of β-galactosidase ( 79), and α-galactosidase ( 80). In some strains of mice there is a time in development at which enzyme activity begins an abrupt decline relative to wild type levels. Each tissue does this at a different time; in liver it begins at 12-15 days of age. This change in enzyme programming is determined by a single locus, Gut, that maps in very close proximity to the structural gene ( 78). The Gut allele producing low activity is invariably associated with the Gush structural allele. The combination results in low heat sensitive activity and has been used as a histochemical marker for genetically distinct cells in chimeras ( 81, 82). Heterozygotes show additive inheritance, but there is contradictory evidence as to whether Gut acts cis or trans ( 83, 84). The Gut site determines developmental patterns prenatally as well as postnatally. Within a few days after fertilization there is a dramatic increase in the β-glucuronidase activity of individual embryos, and the extent and timing of this increase differ in mice carrying various alleles of Gut ( 81).
Recent studies by Gaschow ( 86) indicate that the Gut site, like Gur, controls the rate of enzyme synthesis. Pulse labeling studies have shown that abrupt changes in rates of enzyme synthesis determined by Gut are the cause of the developmental changes in enzyme activity.
A proximate temporal element has also been postulated for the regulation of aryl sulfatase development ( 87). Another, whose location is not known, appears to affect three enzymes in the pyrimidine degradation pathway ( 88).
A temporal locus has been found for α-galactosidase, but in this case it is not linked to the structural gene. Nevertheless, it shows additive inheritance. The α-galactosidase structural gene is on the X chromosome of the mouse ( 89), and the temporal gene specifically determining the liver developmental pattern of α-galactosidase is autosomal ( 80).
Both proximate and distant temporal elements participate in programming the developmental pattern of β-galactosidase levels in liver. C57BL/6 mice have an enzyme development curve that parallels that of other strains in most tissues. However, in liver it begins to deviate at 25 days of age and steadily increases its enzyme activity until in adult life it has twice the level that would otherwise be expected. Crosses between strains carrying alleles of the systemic regulator Bgs showed that a single locus determines this difference and is closely linked to the β-galactosidase complex on chromosome 9 that contains Bgs and Bge ( 90). However, examination of other crosses and recombinant inbred lines indicates that in some cases the failure to undergo this timed elevation in liver is determined by genetic factors unlinked to the β-galactosidase complex on chromosome 9 ( 91). These facts are illustrated by examining the developmental phenotypes of C57BL/6 and C57BL/10 congenic strains carrying various substitutions of the β-galactosidase complex on chromosome 9( Table 2). The rise in liver is absent in the congenic carrying a β-galactosidase complex derived from CBA, which lacks the elevation seen in C57BL/6. On the contrary, the rise is present in the congenic carrying an insertion of the β-galactosidase complex from BALB/c, which also lacks the C57BL/6 liver elevation. Thus, the genetic factors preventing developmental elevation of liver enzyme were carried along with the β-galactosidase complex when it came from CBA, but not when it came from BALB/c.
The H-2 system was defined by Boubelik et al. ( 92) and contains an additional complexity in its genetic structure. H-2 antigen is present on all cells during fetal development except erythrocytes. H-2 antigen appears on erythrocytes after birth; this may occur immediately after birth (the early phenotype) or it may come approximately four days later (the late phenotype). The timing of appearance is not related to the act of birth, but rather to the process of erythrocyte differentiation itself, since the same differences in timing are seen in the erythrocytes that appear after marrow transplants are made into irradiated hosts. In this case the timing of appearance is determined by the donor genotype.
A genetic model was developed from examination of various recombinants and congenic lines and from the segregation of phenotypes during the establishment of five recombinant inbred lines derived from a cross between strains with early and late phenotypes. The model suggests a temporal element, Int, that is in close proximity to the D end of the H-2 region, and acts cis in determining timing control. Int has been separated from H-2, and in recombinants selected between H-2D and the nearby marker Tla segregated with Tla. A second timing system is present that contains two genes Rec and Tem. Tem carries early and late timing alleles, and segregation at Rec determines whether these can override Int. If Rec and Int alleles match then override occurs, if they do not match override does not occur. In order to account for the results it is necessary to assume either that Rec and Tem interact in a cis fashion, although they are 20 centimorgans apart, or more simply, that Rec and Tem are on the X chromosome and thus show haploid expression.
The first major finding in the study of temporal genes is the existence of both proximate and distant sites determining developmental patterns of specific proteins. Second, it is probable that the proximate sites are cis-acting; the evidence for this is good in the H-2 system and for proximate temporal genes seen in other organisms ( 77). Third, the distant sites show additive inheritance when examined quantitatively. Fourth there appear to be specific interactions between the proximate and distant sites, these are seen in the H-2 and β-galactosidase systems in mice and also in the amylase system in Drosophila. An obvious possibility is that proximate sites are the receptors for signals transmitted by the distant sites. If there are signals, the fact that regulation is dosage dependent has strong implications regarding possible mechanisms. Fifth, the relative simplicity of these genetic systems is suggested by the high proportions of all mutants discovered that are proximate sites. Sixth, the systems are relatively specific; the temporal genes determining programs for each enzyme segregate independently of each other even when the programs determine the coordinate regulation of enzymes in development.
Perhaps the major conclusion we can draw is that inbred mouse strains and their derivatives, recombinant inbred lines and congenic strains, have proved to be a very powerful tool for examining questions of gene regulation and cell biology in higher eukaryotes, and especially mammals. The pioneers we honor today built very well indeed. Without the strains they developed it would have been impossible to attack these questions with any measure of success. Success has also depended on the extensive natural polymorphism for regulatory mechanisms that must exist in the wild, and that was frozen in the inbred lines when they were first established, providing the basic material for research.
This extensive variation must have evolutionary significance. It appears that regulatory elements may be as polymorphic as structural genes have been shown to be in recent years. In this context it is significant that so much regulatory information for an enzyme proves to be linked to the structural gene. Indeed, what appears to be segregating in natural populations are gene complexes, chromosome regions whose DNA sequences contain both a structural gene and much associated regulatory information. The existence of such complexes raises the important question of how these two kinds of information are organized in relation to each other.
From the frequency with which regulatory information turns out to be closely linked to the structural gene it is reasonable to assume that a very high proportion of all regulatory information is so linked. We assume that selective forces must exist to maintain this close linkage. The most obvious would be the advantage derived from co-selection of compatible combination of regulatory elements, including some that are necessarily linked to the structural gene for the regulation of transcription. Linkage disequilibrium may well be a reflection of the mechanisms developed to maintain this close association. From this standpoint it is interesting to consider that to whatever extent the extensive natural polymorphism for structural variants is maintained by natural selection, selection is probably operating on the linked regulatory information rather than on the enzyme structure per se. This would be true whether the same base sequences determine both regulation and structure or whether they are determined by separate sequences.
Looking at the total genetic variation in the realization of enzyme activity, it is surprising how much involves posttranslational processing mechanisms rather than regulation of enzyme synthesis. Processing genes are not linked to the enzyme structural gene and generally show recessive/dominant inheritance, in contrast to the regulatory information influencing enzyme synthesis, much of which is closely linked and all of which shows additive inheritance. An interesting point is that additive inheritance can only be seen because enzyme levels are not self-regulated. Mammals, unlike microorganisms, lack a sensing mechanism to determine what enzyme levels are actually present and adjust their regulatory mechanisms accordingly. Thus, heterozygotes for enzyme deficiencies typically have intermediate levels of enzyme. Taken together, the absence of sensing mechanisms in regulating enzyme levels and the presence of additive regulatory information strongly imply that the mechanisms utilized in mammals are not similar to the repressor-like mechanisms seen in microorganisms. It appears that we are, in fact, looking at novel modes of regulation whose analysis is our future challenge.
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