Comparison of nucleotide sequences of mRNAs belonging to the mouse H-2 multigene family

The complete nucleotide sequences of three cDNAs coding for the C-terminal part of mouse histocompatibility (H-2) antigens, and for the 3' non coding regions of these clones have been determined. Comparison of the sequence indicates a large homology throughout the coding and non-coding regions and suggests the existence of a genetic mechanism which homogenizes nucleotide sequences among genes of the H-2 multigene family.     

Identification of members of the P-glycoprotein multigene family.

Overproduction of P-glycoprotein is intimately associated with multidrug resistance. This protein appears to be encoded by a multigene family. Thus, differential expression of different members of this family may contribute to the complexity of the multidrug resistance phenotype. Three lambda genomic clones isolated from a hamster genomic library represent different members of the hamster P-glycoprotein gene family. Using a highly conserved exon probe, we found that the hamster P-glycoprotein gene family consists of three genes. We also found that the P-glycoprotein gene family consists of three genes in mice but has only two genes in humans and rhesus monkeys. The hamster P-glycoprotein genes have similar exon-intron organizations within the 3' region encoding the cytoplasmic domains. We propose that the hamster P-glycoprotein gene family arose from gene duplication. The hamster pgp1 and pgp2 genes appear to be more closely related to each other than either gene is to the pgp3 gene. We speculate that the hamster pgp1 and pgp2 genes arose from a recent gene duplication event and that primates did not undergo this duplication and therefore contain only two P-glycoprotein genes.     

Immunohistochemical localization of acyl-CoA hydrolase/thioesterase multigene family members to rat epithelia

Acyl-CoA hydrolases cleave acyl-CoA thioesters to free fatty acids and coenzyme A. The potency of these enzymes may serve to modulate cellular levels of acyl-CoAs to affect various cellular functions, including lipid metabolism. In this study, we investigated the tissue distribution of this multigene family of enzymes, focusing on cytosolic (CTE-I) and mitochondrial acyl-CoA thioesterases (MTE-I) in adult rats, using an anti-CTE-I antibody which recognizes both the isoforms. Western blotting detected them mainly in organs closely related to fatty acid oxidation, of which kidney contained the highest levels of both enzymes. Immunohistochemistry localized the enzymes primarily in the proximal tubules, where a large energy demand is expected and fatty acids represent a major fuel, correlating well with the intrarenal distribution of peroxisomal beta-oxidation. In situ hybridization suggested colocalization of CTE-I and MTE-I in the kidney. The immunoreactivity was also found in various epithelial tissues in the body, including Harderian gland and sebaceous gland. These results demonstrated the distribution of CTE-I and MTE-I in a wide variety of rat tissues, primarily characterized by an epithelial localization, being consistent with their involvement in fatty acid metabolism.     

Three cDNA sequences of mouse type I keratins. Cellular localization of the mRNAs in normal and hyperproliferative tissues

We have constructed cDNA libraries with poly(A)+ RNA from normal mouse footpad epidermis and from a squamous cell carcinoma of mouse back skin. Both libraries were screened for type I keratin clones. We present sequence data of three keratin cDNA clones which selected mRNAs coding for two 52-kDa proteins (clones pke 52 and pkSCC 52) as well as for a 50-kDa protein (clone pkSCC50). According to their carboxyl-terminal sequences, the two 52-kDa keratin proteins belong to a group of keratins with serine-rich subdomains adjacent to the alpha-helix, whereas the short carboxyl-terminus of the 50-kDa protein lacks a distinct substructure. Sequentially the two 52-kDa keratins are more closely related to each other than to any other mouse type I keratin. However, in situ hybridization with specific subclones reveals a distinctly different pattern of expression in mouse epithelia. Clone pkSCC 52 contains sequence information for a 52-kDa keratin present in basal cells of epidermis and other stratified epithelia, whereas the pke 52 cDNA encodes a keratin which is predominantly expressed in suprabasal cells of nonepidermal tissues. In terms of nucleotide sequence identities, it cannot precisely be decided which of the two mouse 52-kDa proteins is the equivalent of the human epidermal 50-kDa keratin protein (Hanukoglu, I., and Fuchs, E. (1982) Cell 31, 243-252). In the case of the bovine keratin VII, however (Jorcano, J.L., Rieger, M., Franz, J.K., Schiller, D.L., Moll, R., and Franke, W.W. (1984) J. Mol. Biol. 179, 257-281) the sequence identity values speak for an equivalence with the mouse ke 52 keratin. Obviously, in situ hybridization experiments would best be suited to unravel the precise interspecies relationship between the four highly similar keratins. The discriminatory efficacy of this technique is further emphasized by the demonstration that the mRNA for a 50-kDa keratin is present not only in hyperproliferative epithelia, but also in normal cells of hair follicles.     

Detecting and characterizing gene conversions between multigene family members

We used a variety of methods to detect known gene conversions in the actin gene families of five angiosperm species, the beta-globin gene families of two primate species, and the Zfx/Zfy gene families of seven mammalian species. Our goal was to devise a working strategy which would allow the analysis of the members of a multigene family in order to determine whether there had been gene conversions between its members, identify the genes involved in the gene conversions, establish the lengths of the converted regions, and determine the polarities of the gene conversions. We show that three phylogenetic methods and the homoplasy test of Maynard Smith and Smith perform relatively poorly on our data sets because the sequences we analyzed had large levels of multiple substitutions. The method of Sawyer, the compatibility method of Jakobsen and Easteal, the partition matrix method of Jakobsen, Wilson, and Easteal, and the co-double method of Balding, Nichols, and Hunt can be used to identify the genes which have been involved in gene conversions. The co-double method is more powerful than other methods but requires orthologous sequences from related species. Compatibility, phylogenetic, and nucleotide substitution distribution statistics methods can be used to identify the location of the converted region(s). Site-by-site compatibility analyses can also be used to identify the direction of the conversion event(s). Combinations of these methods can therefore be used to establish the presence, locations, and polarities of gene conversions between multigene family members.     

Sequence of rat alpha- and gamma-casein mRNAs: evolutionary comparison of the calcium-dependent rat casein multigene family.

The complete sequences of rat alpha- and gamma-casein mRNAs have been determined. The 1402-nucleotide alpha- and 864-nucleotide gamma-casein mRNAs both encode 15 amino acid signal peptides and mature proteins of 269 and 164 residues, respectively. Considerable homology between the 5' non-coding regions, and the regions encoding the signal peptides and the phosphorylation sites, in these mRNAs as compared to several other rodent casein mRNAs, was observed. Significant homology was also detected between rat alpha- and bovine alpha s1-casein. Comparison of the rodent and bovine sequences suggests that the caseins evolved at about the time of the appearance of the primitive mammals. This may have occurred by intragenic duplication of a nucleotide sequence encoding a primitive phosphorylation site, -(Ser)n-Glu-Glu-, and intergenic duplication resulting in the small casein multigene family. A unique feature of the rat alpha-casein sequence is an insertion in the coding region containing 10 repeated elements of 18 nucleotides each. This insertion appears to have occurred 7-12 million years ago, just prior to the divergence of rat and mouse.     

Expression of members of the Saccharomyces cerevisiae hsp70 multigene family

The hsp70 multigene family of Saccharomyces cerevisiae is a complex multigene family, composed of members exhibiting complex patterns of regulation. Expression of some members is induced after a heat shock, whereas expression of others is repressed. Some members of the family are expressed during exponential growth. One gene, SSA3, shows an unusual pattern of expression during approach to stationary phase. While most RNAs decrease in abundance, SSA3 RNA levels dramatically increase. The constitutive expression of SSA3 in cells lacking adenylate cyclase activity suggests that cAMP modulates SSA3 expression.     

Evolution of the casein multigene family: conserved sequences in the 5'' flanking and exon regions.

The rat alpha- and bovine alpha s1-casein genes have been isolated and their 5' sequences determined. The rat alpha-, beta-, gamma- and bovine alpha s1-casein genes contain similar 5' exon arrangements in which the 5' noncoding, signal peptide and casein kinase phosphorylation sequences are each encoded by separate exons. These findings support the hypothesis that during evolution, the family of casein genes arose by a process involving exon recruitment followed by intragenic and intergenic duplication of a primordial gene. Several highly conserved regions in the first 200 base pairs of the 5' flanking DNA have been identified. Additional sequence homology extending up to 550 base pairs upstream of the CAP site has been found between the rat alpha- and bovine alpha s1-casein sequences. Unexpectedly, the 5' flanking promoter regions are conserved to a greater extent than both the entire mature coding and intron regions of these genes. These conserved 5' flanking sequences may contain potential cis regulatory elements which are responsible for the coordinate expression of the functionally-related casein genes during mammary gland development.     

Differential localization of distinct keratin mRNA-species in mouse tongue epithelium by in situ hybridization with specific cDNA probes

The tongue of the adult mouse is covered by a multilayered squamous epithelium which is continuous on the ventral surface, however interrupted on the dorsal surface by many filiform and few fungiform papillae. The filiform papillae themselves are subdivided into an anterior and posterior unit exhibiting different forms of keratinization. Thus, the entire epithelium shows a pronounced morphological diversity of well recognizable tissue units. We have used a highly sensitive in situ hybridization technique to investigate the differential expression of keratin mRNAs in the tongue epithelium. The hybridization probes used were cDNA restriction fragments complementary to the most specific 3'-regions of any given keratin mRNA. We could show that independent of the morphologically different tongue regions, all basal cells uniformly express the mRNA of a type I 52-kD keratin, typical also for basal cells of the epidermis. Immediately above the homogenous basal layer a vertically oriented specialization of the keratin expression occurs within the morphological tissue units. Thus the dorsal interpapillary and ventral epithelium express the mRNAs of a type II 57-kD and a type I 47-kD keratin pair. In contrast, in the anterior unit of the filiform papillae, only the 47-kD mRNA is present, indicating that this keratin may be coexpressed in tongue epithelium with different type II partners. In suprabasal cells of both, the fungiform papillae and the posterior unit of the filiform papillae, a mRNA of a type I 59-kD keratin could be detected; however, its type II 67-kD epidermal counterpart seems not to be present in these cells. Most surprisingly, in distinct cells of both types of papillae, a type I 50-kD keratin mRNA could be localized which usually is associated with epidermal hyperproliferation. In conclusion, the in situ hybridization technique applied has been proved to be a powerful method for detailed studies of differentiation processes, especially in morphologically complex epithelia.     

Comparison of class I (H-2) gene sequences. Derivation of unique probes for members of this multigene family

The genes for the classical transplantation antigens are unique in that they belong to a multigene family of which each member is represented by a large number of alleles. Since all of these genes are highly related in sequence, it has been difficult to study the expression of individual members of this complex gene family. Based upon our initial suggestion that the 3' noncoding regions of these genes may be useful in identifying mRNA molecules transcribed from different loci, we have compared a large number of sequences from different inbred mouse strains and have been able to assign each of these sequences without ambiguity into distinct allelic series. Such accurate assignment has afforded the opportunity to compare the coding regions of these highly homologous genes and has led to the identification of sequences which are apparently unique to specific genes in the family. Synthetic oligonucleotides corresponding to each of the locus-specific unique regions have been used successfully to type a panel of cDNA sequences, as well as to quantitate the relative amounts of mRNA transcribed from distinct loci. The availability of these specific coding probes will allow the analysis of individual genes and their specific expression without interference from other highly homologous sequences in this multigene family.     

Isolation of a multigene family containing human alpha-tubulin sequences

The boundaries of the origin of polyoma DNA replication have been analyzed using a set of deletion mutants. The majority of these had small deletions, 5 to 30 base-pairs in size, which together removed most of the non-translated sequences of the genome. The phenotype of the mutants was characterized by analysis of infectivity, transforming ability and DNA synthesis. All mutants with reduced or abolished infectivity had corresponding defects of viral DNA synthesis. The effect of the deletion was cis-acting, since the replication of the mutants was not stimulated by the presence of wild-type DNA. Deletions causing a reduction of DNA synthesis were found at two sites. The first at the 32 base-pair inverted repeat sequence and the neighbouring A · T tract previously implicated in the initiation of DNA synthesis, and the second close to the late genes. The two sites were separated by at least 60 base-pairs of non-essential DNA. Only one mutant with a deletion at the second site was unable to express early gene functions.The mutants were constructed by linearization, shortening and recircularization of polyoma DNA inserted into the plasmid pBR322. The mutagenesis was directed at restriction endonuclease BglI or PvuII cleavage sites. The BglI-directed mutagenesis was focussed to polyoma DNA by using as a vector a derivative of pBR322 resistant to cleavage by BglI.     

The mutational load of a multigene family with uniform members

The mutational load of a multigene family with uniform members was studied by computer simulations. Two models of selection, truncation and exponential fitness, were examined, by using a simple model of gene conversion. It was found that the load is much smaller than the Haldane-Muller prediction under the truncation selection, and that it becomes approximately equal to the value calculated by the formula, nv(1-q)/(m-nq), where n is the copy number, v is the rate of detrimental mutation per gene copy, m is the truncation point in terms of the number of detrimental genes eliminated, and q is the equilibrium frequency of detrimental mutation. However the equilibrium frequency cannot be analytically obtained. For the exponential fitness model, the load is close to the Haldane-Muller value. When there is no gene conversion, the load becomes larger than the cases with conversion both for the truncation and the exponential fitness models. Thus, gene conversion or other mechanisms that are responsible for contraction-expansion of mutants on chromosomes helps eliminating deleterious mutations occurring in multigene families.     

Isolation of distantly related members in a multigene family using the polymerase chain reaction technique.

We have designed a strategy to isolate and identify genes (cDNAs) coding for distantly-related members within a large multigene family. We have used limited protein sequence information data to delineate conserved regions where members of a supergene family are related. Comparison of the nucleotide sequences of such conserved areas defined consensus sequences that were used for the synthesis of deoxynucleotide primers. Two forward and two reverse primers were synthesized, and four separate pairs of primer combinations were used under low stringency in polymerase chain reactions (PCR) to generate amplified DNA products. The PCR products were directionally cloned into the phage vector M13mp18. Each of four libraries was screened with radiolabeled PCR product generated using a pair of primers different from those used to generate the library. Using this approach on the supergene family of ligand-gated ion channels, we were able to isolate and identify two novel subunits of neurotransmitter-operated ion channels.     

Isolation of a murine Ly-6 cDNA reveals a new multigene family.

The Ly-6 alloantigens have been shown to play a critical role in T lymphocyte activation. To isolate a Ly-6 cDNA, synthetic oligonucleotides, based on the partial amino acid sequence of purified Ly-6E.1 protein, were used to probe a cDNA library. The synthetic oligonucleotides or the isolated cDNA detected a 1.1-kb RNA species. Sequence analysis of the cDNA clone revealed that the Ly-6E.1 protein consists of a 26-amino acid leader followed by a 108-residue, cysteine-rich, core protein with no N-linked glycosylation sites. Southern blot analysis of genomic DNAs revealed multiple bands indicating a family of related genes. Using recombinant inbred and Ly-6 congenic strains of mice, restriction fragment length polymorphisms were demonstrable, and correlated with the Ly-6 allotype of the DNA donors. This probe will enable further molecular genetic analysis of the role of Ly-6-linked proteins in the process of T lymphocyte activation. Isolation of Ly-6 genomic clones may promote a further understanding of the complex tissue-specific expression patterns characteristic of Ly-6-linked genes.     

In situ localization of PCR-amplified DNA and cDNA

Combining the high sensitivity of PCR with the cell localizing ability ofin situ hybridization allows for the reproducible detection of low copy targets in intact cells. This article describes several key variables that include fixation, protease digestion, the hot start maneuver, stringency, and, for RNA analysis, DNase digestion that are important to successfulin situ PCR. Also stressed is the importance of performing and interpreting controls with each experiment. Important controls include omission of key components, use of samples known either to contain or lack the target of interest and, most importantly, the in-built controls invariably present in the heterogeneous component of any given tissue type.