The UCP2-UCP3 gene cluster maps to chromosome 11q13 in humans, and polymorphisms in these genes may contribute to obesity through effects on energy metabolism. DNA sequencing of UCP2 and UCP3 revealed three polymorphisms informative for association studies: an Ala-->Val substitution in exon 4 of UCP2, a 45 bp insertion/deletion in the 3'-untranslated region of exon 8 of UCP2 and a C-->T silent polymorphism in exon 3 of UCP3. Initially, 82 young (mean age = 30 +/- 7 years), unrelated, full-blooded, non-diabetic Pima Indians were typed for these polymorphisms by direct sequencing. The three sites were in linkage disequilibrium ( P < 0.00001). The UCP2 variants were associated with metabolic rate during sleep (exon 4, P = 0.007; exon 8, P = 0.016) and over 24 h (exon 8, P = 0.038). Heterozygotes for UCP2 variants had higher metabolic rates than homozygotes. The UCP3 variant was not significantly associated with metabolic rate or obesity. In a further 790 full-blooded Pima Indians, there was no significant association between the insertion/deletion polymorphism and body mass index (BMI). However, when only individuals >45 years of age were considered, heterozygotes (subjects with the highest sleeping metabolic rate) had the lowest BMI (P = 0.04). The location of the insertion/deletion polymorphism suggested a role in mRNA stability; however, it appeared to have no effect on skeletal muscle UCP2 mRNA levels in a subset of 23 randomly chosen Pima Indians. In conclusion, these results suggest a contribution from UCP2 (or UCP3) to variation in metabolic rate in young Pima Indians which may contribute to overall body fat content later in life.

An autosomal genomic scan to search for linkage to obesity and energy metabolism was completed in Pima Indians, a population prone to obesity. Obesity was assessed by percent body fat (by hydrodensitometry) and fat distribution (the ratio of waist circumference to thigh circumference). Energy metabolism was measured in a respiratory chamber as 24-h metabolic rate, sleeping metabolic rate, and 24-h respiratory quotient (24RQ), an indicator of the ratio of carbohydrate oxidation to fat oxidation. Five hundred sixteen microsatellite markers with a median spacing of 6.4 cM were analyzed, in 362 siblings who had measurements of body composition and in 220 siblings who had measurements of energy metabolism. These comprised 451 sib pairs in 127 nuclear families, for linkage analysis to obesity, and 236 sib pairs in 82 nuclear families, for linkage analysis to energy metabolism. Pointwise and multipoint methods for regression of sib-pair differences in identity by descent, as well as a sibling-based variance-components method, were used to detect linkage. LOD scores >=2 were found at 11q21-q22, for percent body fat (LOD=2.1; P=.001), at 11q23-q24, for 24-h energy expenditure (LOD=2.0; P=.001), and at 1p31-p21 (LOD=2.0) and 20q11.2 (LOD=3.0; P=.0001), for 24RQ, by pointwise and multipoint analyses. With the variance-components method, the highest LOD score (LOD=2.3 P=.0006) was found at 18q21, for percent body fat, and at 1p31-p21 (LOD=2.8; P=.0003), for 24RQ. Possible candidate genes include LEPR (leptin receptor), at 1p31, and ASIP (agouti-signaling protein), at 20q11.2.

Because tumor necrosis factor-a (TNF-a) expression is increased in adipose tissue of both rodent models of obesity and obese humans, it has been considered as a candidate gene for obesity. Pima Indians were scored for genotypes at three polymorphic dinucleotide repeat loci (markers) near the gene TNF-a at 6p21.3. In a sib-pair linkage analysis, percent body fat, as measured by hydrostatic weighing, was linked (304 sib-pairs, P = 0.002) to the marker closest (10 kb) to TNF-a. The same marker was associated (P = 0.01) by analysis of variance with BMI. To search for possible DNA variants in TNF-a that contribute to obesity, single stranded conformational polymorphism analysis was performed from 20 obese and 20 lean subjects. Primer pairs were designed for the entire TNF-a protein coding region and part of the promoter. Only a single polymorphism located in the promoter region was detected. No association could be demonstrated between alleles at this polymorphism and percent body fat. We conclude that the linkage of TNFa to obesity might be due to a sequence variant undetected in TNF-a or due to a variant in some other closely linked gene (J. Clin. Invest. 1995.96:158-162.)

The homologues of single genes that cause obesity in rodents are suggested as candidate genes for modulation of body composition in humans. Among these genes are the four mouse mutations-diabetes (db), obesity (ob), tubby (tub), and yellow agouti (Ay). Variation in the human counterparts to these genes (OB, DB, TUB, and ASP, respectively) may contribute to human obesity, which is thought to have a substantial genetic component. To initially assess the potential contribution of these genes to human obesity, we examined polymorphic DNA markers that, by virtue of syntenic relationships to appropriate regions of the mouse genome, should be closely linked to the human counterparts of these genes. Using combined data from 716 Pima Indians comprising 217 nuclear families, we have tested a number of polymorphic microsatellite markers (three at DB, two at OB, five at TUB, and three at ASP) for sib-pair linkage to BMI, percentage body fat, resting metabolic rate, 24-h energy expenditure, and 24-h respiratory quotient. No significant linkages were found in an analysis of all sibships or in an analysis restricted to discordant sib pairs.

In Drosophila pseudoobscura, the amylase (Amy) multigene family is contained within a series of inversions, or gene arrangements, on the third chromosome. The Standard (ST), Santa Cruz (SC), and Tree Line (TL) inversions are central to the phylogeny of arrangements, and have clusters of other arrangements derived from them. The gene arrangements belonging to each of these three clusters have a characteristic number of Amy genes, ranging from three in ST to two in SC to one in TL. This distribution pattern can reflect a history of either duplications or deletions, although the data available in the past did not permit a decision between these alternatives. We provide unambiguous evidence that three Amy genes were present before the divergence of the ST, SC, and TL arrangements. Thus, the current status of the Amy multigene family is the result of deletions in the TL and SC arrangements, which created three new pseudogenes: TL Amy2-psi, TL Amy3-psi, and SC Amy3-psi. Analysis of pseudogene sequences revealed that, in the SC and ST arrangements, pseudogene evolution has been retarded, most likely due to the homogenization effect of gene conversion. Finally, by determining the original copy number, we have reconstructed the evolutionary history of the Amy multigene family and linked it with the evolution of the central gene arrangements.

The Amylase locus in Drosophila melanogaster normally contains two copies of the structural gene for alpha-amylase, a centromere-proximal copy, Amy-p, and a distal copy, Amy-d. Products of the two genes may display discrete electrophoretic mobilities, but many strains known to carry the Amy duplication are characterized by a single amylase electromorph, e.g., Oregon-R, which produces the mobility variant AMY-1. A transient expression assay was used in somatic transformation experiments to test the functional status of the Amy genes from an Oregon-R strain. Plasmid constructs containing either the proximal or distal copy were tested in amylase-null hosts. Both genes produced a functional AMY-1 isozyme. Constructs were tested against an AMY-3 reference activity produced by a coinjected plasmid that contains the Amy-d3 allele from a Canton-S strain. With reference to the internal control, the Amy-p and Amy-d genes from Oregon-R expressed different relative activity levels for AMY-1 in transient assays. The transient expression assay was successfully used to test the functional status of Amy-homologous sequences from strains of other species of Drosophila characterized by a single amylase elctromorph, namely, Drosophila pseudoobscura ST and Drosophila miranda S 204. The amylase-null strain of D. melanogaster provided the hosts for these interspecific somatic transformation experiments.

The functional locus for alpha-amylase (Amy) in Drosophila miranda is in the evolutionarily new X2 chromosome. X2 evolved from an autosome in response to an ancestral autosome-Y translocation that gave rise to the "neo-Y" chromosome of this species. Y-linked Amy, if still present in the ancestrally translocated element, is unexpressed. Dosage compensation for amylase activity was examined in larvae of the S 204 strain. Since dietary glucose is known to repress Amy expression in Drosophila melanogaster, dosage compensation of amylase activity in male larvae of D. miranda was tested by rearing larvae of both sexes on yeast diets with or without a glucose supplement. The WT 10 strain of Drosophila persimilis, a sibling species in which Amy is autosomally linked, was used as a reference for tests of amylase activity differences between the sexes. On the diet with glucose, Amy expression was repressed in both WT 10 and S 204 larvae and male larvae of S 204 displayed dosage compensation for amylase activity. On the nonrepressing diet consisting of yeast alone, S 204 continued to display dosage compensation.

The Amylase locus in Drosophila melanogaster contains duplicate, divergently transcribed structural genes for alpha-amylase, AmyA and AmyB. A sensitive and reliable transient expression assay was developed for testing amylase activities produced by exogenous Amy genes in somatically transformed larvae of an amylase-null strain of flies. Alleles tested, AmyA and AmyB, came from recombinant clone lambda Dm65, which contains genomic DNA from a Canton-S strain. The transient assay was used in a deletion analysis aimed at locating cis-regulatory sequences within the 5' region of AmyB. Results suggest that upstream regulatory sequences for correct spatial expression of AmyA and AmyB in third-instar larvae are located within 446 and 430 bp of their respective starts for transcription. A sequence required for high levels of AmyB expression was located within its 5' upstream region between the base pairs at -332 and -219. AmyA does not appear to have a comparable regulatory element in its 5'-flanking sequence. Barely detectable expression of AmyB was observed when it was flanked by only 92 bp of upstream sequence. A model is proposed for incomplete coordinate control of the duplicate Amy genes.