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Previously we found that male mice carrying either of two attenuated herpes simplex virus thymidine kinase reporter transgenes displayed low level ectopic expression of the reporter gene in the testis and, although fertile, exhibited reduced fecundity. In contrast to males of later generations, many of the founder males failed to transmit the transgene to their progeny. This led to the suggestion that these fertile non-transmitting males are mosaic, with the sperm developing from the non-transgenic lineage outperforming those from the heterozygous transgenic lineage. Here we present the results of artificial insemination (AI) and in vitro fertilization (IVF) experiments designed to test this hypothesis. Albino CF(1) hybrid females were inseminated with mixtures of equal numbers of sperm from heterozygous transgenic (HT) males (equivalent to C57BL/6 x CBAF(2)) and CF(1) males. Similar mixed inseminations were carried out in parallel with sperm from non-transgenic (NT) siblings of the HT mice and 13-day fetuses were scored by eye color to determine their paternity. The pooled data from five experiments gave ratios of CF(1) to HT and CF(1) to NT offspring of 8.13 and 0.22 respectively, implying a calculated HT to NT ratio of 0.027. This indicates that, in competition with each other, the NT sperm would be almost 40 times more successful in fertilization than the HT sperm. Smaller differences were observed between HT and NT when AI was performed with unmixed sperm, consistent with the fertility of HT non-founder males. However, in five IVF experiments carried out with unmixed sperm, 142/212 oocytes exposed to NT sperm were activated and divided, while only 8/226 oocytes treated with HT sperm reached the two-cell stage. This confirms that HT sperm are defective and indicates that the IVF method employed amplified these deficiencies, which may have only a small effect upon natural reproduction when the HT sperm are not in competition with normal sperm.

The main route and, in most species, the only reliable route to the generation of transgenic animals is by microinjecting DNA into an early embryo, generally one of the pronuclei of a newly fertilized egg (a one-cell embryo). In most cases, a small number (perhaps 100) of identical cloned DNA molecules is introduced in this way. The weight of evidence supports the view that this DNA forms extrachromosomal concatemers (arrays), mainly of monomers orientated in the same direction, by rounds of homologous recombination. Since this occurs when a population of identical linear molecules is introduced, productive recombination can only take place after a population of circularly permuted monomers has been generated by circularization and random cleavage. Extrachromosomal recombination is known to occur by a nonconservative process in transfected mammalian cells in culture. Concatemeric molecules integrate into the chromosomes, more or less at random, by illegitimate recombination. This may occur during DNA replication, consistent with the very high observed frequency of transgenic founder animals that are mosaics of transgenic and nontransgenic cells. Foreign genes integrated in this way are frequently liable to chromosomal position effects, which can adversely affect expression. In the commercial arena this often necessitates the production of a large number of transgenic founders in the hope of obtaining one with a high expression level. Ways of approaching this practical problem are explored.

The multigene family which codes for the mouse major urinary proteins (MUPs) consists of approximately 35 genes. Most of these are members of two different groups, Group 1 and Group 2, which can be distinguished by nucleic acid hybridisation. Here we describe the structure of a Group 1 gene and show that two size classes of MUP mRNA which are found in mouse liver result from different splicing events in the 3'-non-coding region and contain different polyadenylation sites. Short mRNA is approximately 750 nucleotides long, contains six exons, and is the main product of the Group 2 genes. Long mRNA is approximately 880 nucleotides long, contains seven exons and is the main product of the Group 1 genes. Five exons and part of the sixth are common to long and short mRNA and contain the coding region. This codes for an acidic protein of 180 amino acids containing an 18 residue signal peptide. A comparison of the mouse sequence with a homologous rat alpha 2u-globulin sequence shows that the rate of evolutionary divergence of the two proteins has been high. Silent sites have diverged four times more rapidly than replacement sites, showing that there has been selection against change in the protein sequence.

Fourteen different major urinary protein (MUP) genomic clones from BALB/c mice were isolated. By restriction site mapping, six of these form two sets of three overlapping clones. By the criterion of cross-hybridization, the 10 different genes fall into two groups of four (Group 1) and three (Group 2) genes, while three genes fall into neither group. Southern blot analysis of genomic DNA with Group 1 and Group 2 plasmid subclones shows that the haploid mouse (BALB/c) genome contains approximately 15 Group 1 genes, 12 Group 2 genes and at least seven MUP genes that belong to neither group. An analysis of mouse-Chinese hamster hybrid cell lines shows that most, if not all, Group 1 and Group 2 genes are located on mouse chromosome 4.

The family of mouse major urinary protein (MUP) genes has about 35 members, clustered together on chromosome 4. Most of the genes belong to two major subfamilies (group 1 and group 2) each with 12-15 members. Recently we showed that most of the group 1 and group 2 genes are arranged in pairs, each containing a group 1 and a group 2 gene in divergent transcriptional orientation, with 15 kilobases of DNA between the two cap sites. Here we present the nucleotide sequence of the first exon of six group 1 genes and four group 2 genes. The data confirm the close relationship of the genes within each group and the considerable divergence of the two groups from each other. The four group 2 genes all carry the same nonsense mutation in codon 7 of the sequence that specifies the mature protein. Thus, not only do these genes have a common ancestor, but also it seems that their amplification followed the mutation of the ancestor to a pseudogene. Taking into account the 3' flanking regions of the two genes, the overall size of each gene-pair is about 45 kilobases. The sequencing data supports our earlier suggestion that this 45 kilobase domain is the unit of Mup amplification.

Here we compare the exonic sequences of four Group 1 mouse major urinary protein (MUP) genes and four Group 1 cDNA sequences. These define seven different nucleotide sequences which differ from each other by 0.35% of bases on average, and which would code for seven different MUP proteins that could probably be resolved physically into at least five classes. The sequences differ at 13 nucleotide positions and at six codons, and although they are closely related their descent cannot be described by a simple series of duplications. We also describe the sequence of another liver cDNA (pMUP15) which has diverged from the Group 1 consensus sequence in 14.6% of bases. The divergence is much greater over exons 1-3 than over exons 4-6, suggesting that an ancestral gene conversion event has occurred. pMUP15 also differs from the Group 1 genes in having a longer signal peptide sequence and a different splice configuration between exons 6 and 7. Unlike the Group 1 sequences, pMUP15 contains a potential N-linked glycosylation site. Other published work has shown that a shorter cDNA clone which is identical over their common sequence to pMUP15 codes for MUP proteins that are unusually large in size and acidic in pI. We show here that mouse urine does indeed contain a glycosylated MUP protein with those properties, presumably the product of the gene that corresponds to pMUP15.

The multigene family which codes for the mouse major urinary proteins consists of about 35 genes. Most of these are members of two distinct groups, group 1 and group 2. The group 1 and group 2 genes are organized in head-to-head pairs within 12 to 15 remarkably uniform chromosomal units or domains about 45 kilobase pairs (kb) in size. The 45-kb units are located on chromosome 4, and many of them are adjacent to each other. We propose that the 45-kb unit is a unit both of organization and of evolutionary change. In this study the homologies within the unit were observed by examining, in an electron microscope, heteroduplex and foldback structures made from cloned major urinary protein genes. These show that the 45-kb unit is a gigantic imperfect palindrome. Each arm of the palindrome contains two regions of inverted symmetry of 9.5 and 4.5 kb separated by a 3-kb nonsymmetrical region. We argue that the nonsymmetrical regions arose by a series of deletion events in the two arms of the palindrome. The center of the 45-kb unit is an 8-kb sequence without inverted symmetry flanked by the 9.5-kb regions, which contain the 4-kb genes and their immediate 5' and 3' flanking regions. The junction between adjacent 45-kb units is a 2- to 4-kb sequence without inverted symmetry flanked by the 4.5-kb regions. Some of the 45-kb units are arranged as direct tandem repeats. Others appear to be in inverted orientation with respect to a neighboring unit. Cloned major urinary protein genes show few incidences of the repetitive elements B1, B2, R, and MIF. Two elements, a B1 and an R, may be a constant feature of the 45-kb units. If so, in those cases in which the units are in tandem array, both of these elements will occur with a 45-kb periodicity. A comparison of corresponding parts of different 45-kb units shows that they differ because of a number of deletion or insertion events, particularly in the regions 3' to the genes.

Histidine decarboxylase was purified 800-fold from the kidneys of thyroxine-treated mice. The purification procedure included precipitation of protein from a crude supernatant after heating it to 55 degrees C at pH 5.5, fractionation with (NH4)2SO4, phosphocellulose column chromatography, chromatofocusing, DEAE-Sepharose column chromatography, gel filtration on Sephacryl S-300 and preparative polyacrylamide-gel electrophoresis. The native enzyme had an estimated Mr of 113 000. The protein was analysed in SDS/10%-polyacrylamide gels and formed a single band corresponding to a subunit Mr of 55 000, indicating that it is a dimer. Three forms of the enzyme were resolved on isoelectrofocusing gels, with pI 5.3, 5.5 and 5.7.

We describe ten Charon 4A genomic DNA clones from BALB/c mice which include at least seven different major urinary protein (MUP) genes. We have established the orientation of all seven sequences, and have placed six of them in precise register by means of restriction site maps and Southern blot hybridization with cloned cDNA sequences. Four of the seven genomic sequences (family I sequences) form hybrids with six independent cDNA clones that have a high thermal stability and hybridize more strongly with mRNA from three inbred mouse lines. Hybrids between the remaining three genomic sequences and the cDNA clones have a lower thermal stability and hybridize less strongly with mRNA from the three inbred lines. Homologies between different cloned sequences extend over as much as 15 kb. No clone contains parts of two MUP genes, and no homology has been detected between the 3' flanking region of one MUP gene and the 5' flanking region of another.

The genome of the laboratory mouse contains about 35 major urinary protein (MUP) genes, many of which are clustered on chromosome 4. We have used distance and parsimony methods to estimate phylogenetic relationships between MUP genes from nucleotide sequence and restriction maps. By analyzing coding sequences we show that the genes fall into four main groups of related sequences (groups 1–4). Comparisons of restriction maps and the nucleotide sequences of hypervariable regions that lie 50 nucleotides 5′ to the cap sites show that the group 1 genes and probably also the group 2 pseudogenes fall into subgroups. The most parsimonious trees are consistent with the evolution of the array of group 1 and 2 genes by mutation accompanied by a process tending toward homogenization such as unequal crossing-over or gene conversion. The phylogenetic grouping correlates with grouping according to aspects of function. The genomes of the inbred strains BALB/c and C57BL contain different MUP gene arrays that we take to be samples from the wild population of arrays.

We have compared poly(A)-mRNA isolated from mouse liver endoplasmic reticulum (ER) membranes (ER mRNA) with that from free polyribosomes (free mRNA) by translation in a cell-free system and by mRNA--cDNA reassociation analysis. The partitioning of certain translation products between the two fractions suggests that the physical contamination of the free mRNA by the ER is 10%, and contamination of the ER by the free is 1%. Reassociation crossreactions suggest that no sequences are totally absent from either fraction. In particular, the rare sequences are shared between the two fractions. Among the more abundant sequences, we detect three groups: (i) a group of about 10 sequences that are relatively abundant in both fractions; (ii) a group that occurs mostly in the free fraction; (iii) a group of about 12 sequences, 6 or 7 of them encoding secretory polypeptides. The members of this group are largely or entirely confined to the ER fraction. Secretory proteins are almost entirely absent from the products of th free mRNA fraction. Quantitation of individual mRNA sequences by hybridization with cDNA clones shows that a range of sequence concentrations exists within the abundant class of ER mRNA.

A detailed characterization of messenger RNA from a permanent line of rat myoblasts ia reported. Messenger RNA was defined according to several criteria: size distribution, insensitivity of its synthesis to low levels of actinomycin D, polyadenylation and association with polyribosomes. Both pulse-labelled RNA and the steady-state RNA population were investigated. In all cases the mRNA was found to contain a minor fraction complementary to repetitive DNA sequences and a major fraction complementary to sequences that are non-repetitive or nearly so. Low molecular weight ( 18 S) mRNA. The two classes could be separated to a large extent by annealing the mRNA with Sepharose to which repetitive DNA had been bound. The results obtained show that most repetitive sequence transcripts are not covalently linked to non-repetitive sequence transcripts. Approximately 20% of the mRNA population in these cells appears to be transcribed from repetitive sequences. Experiments carried out with complementary DNA transcribed from the mRNA population by using avian myeloblastosis virus reverse transcriptase support these conclusions in general, although there are indications that transcription of mRNA by the enzyme is not random. A substantial fraction of the complementary DNA (up to 25%) forms duplexes with DNA sequences that are very moderately repetitive (less than tenfold). Because of the likelihood that the mRNA is non-randomly transcribed, it is not clear that the same can be said of the mRNA population.

Laboratory mouse strains carry approximately 35 major urinary protein (MUP) genes per haploid genome, tightly clustered together on chromosome 4. Most belong to two main groups (Groups 1 and 2). The available evidence strongly suggests that the Group 1 genes are active while the Group 2 genes are pseudogenes. Here we present the complete sequence of a Group 1 gene and a Group 2 gene and 700 bp of flanking sequence. The sequence of the Group 1 gene is consistent with its being active. The Group 2 gene contains two stop codons and a frame-shift mutation in the reading frame defined by the Group 1 gene, and would code for a signal peptide 25 rather than 19 amino acids long. The Group 2 gene differs from the Group 1 gene in other ways: a deletion upstream of the TATA box and another in intron 3, a base change in the TATA box itself, a 2 bp duplication at the splice acceptor boundary of intron 6, an altered poly(A) addition signal and a 1-base deletion 5' to the initiation codon. Some of these differences may explain the 10- to 20-fold higher level of Group 1 mRNA in mouse liver, and the fact that Group 1 and Group 2 transcripts are mainly spliced differently. The presence of the stop codon means that the Group 2 gene is a pseudogene in the context of the Group 1 gene. However, there is some evidence that the mature hexapeptide that it would code for may have biological activity. The 12 acceptor splice sites of the two genes all contain the identical sequence ACAG at the exon boundary.(ABSTRACT TRUNCATED AT 250 WORDS)

Most transgenic animals have been produced by directly injecting DNA into one of the embryo pronuclei in the period immediately following fertilization. Transgenic animals are produced when DNA becomes integrated into the chromosomes, in most cases the transgenic genotype is transmitted to progeny through the germ line. The characteristics of the foreign DNA include tandem arrangement of multiple copies of the input DNA, predominantly in direct rather than inverted orientation, and illegitimate recombination with the chromosome at the site of integration. Foreign DNA integrated into chromosomes of cultured cells has identical characteristics. We argue that these and other similarities between the integration of foreign DNA into the chromosomes of microinjected embryos on the one hand and on the other of transfected and microinjected cells, strongly suggest that the processes have the same basis. By considering mainly the literature relating to cell transfection and microinjection, we conclude that tandemly arranged concatemers of input foreign DNA are built up by a process of homologous but non-conservative recombination. End-joining and illegitimate recombination events characterize the integration of the concatemers into the chromosome, and also contribute to the formation of the concatemers. We also suggest that these superficially different processes are based on the same opportunistic repair-ligation mechanism; the frequencies of the different types of event reflect both the frequencies with which different sorts of association occur between DNA molecules and their relative stability.

Poly(A)mRNA was isolated from the endoplasmic reticulum (ER) or male BALB/c mice and used as the template for cDNA synthesis. Double-stranded (ds) cDNA was cloned in a bacterial plasmid and 21 clones were selected for further study. Six clones have been identified by hybrid selection of mRNA, translation of the mRNA in a template-dependent system, polyacrylamide gel electrophoresis and specific immunoprecipitation. Four code for the major urinary protein of the mouse (MUP), one for serum albumin and one for α 1 -antitrypsin. A seventh codes for an unientified polypeptide with M r = 27 000. Initial-rate hybridisation kinetics of immobilised cloned sequences with end-labelled mRNA showed that serum albumin mRNA is the most abundant in the population of mRNA species present in the endoplasmic reticulum fraction of female BALB/c liver. MUP mRNA is also very abundant. The α 1 -antitrypsin mRNA and the mRNA specifying the 27 000 dalton protein are about 5 times less abundant than serum albumin mRNA on a molar basis.

The mouse major urinary proteins (MUPs) and the unprocessed in vitro translation products of MUP mRNA were each resolved by isoelectric focusing (IEF). The urinary MUPs showed about 15 distinct components, and the unprocessed MUPs about 20. In each case wide variation was observed in the relative intensities of individual bands. A comparison of three inbred lines (C57BL, BALB/c and JU) showed inter-line variation in the patterns both of the urinary MUPs and of the unprocessed MUPs. A series of experiments was carried out with a cloned MUP cDNA probe. All three inbred lines contain the same number (about 20) of MUP genes per haploid genome. In Southern blot analysis of genomic DNA the MUP genes displayed complex patterns which we interpret as showing variation on a common basic MUP gene sequence. For each combination of restriction enzymes tested, one size of fragment carried more than half of the total label, and this fragment was always the same in the three inbred lines. Inter-line differences were observed in the patterns of some of the less reactive fragments. MUP mRNA consists of at least two distinct species with sizes of 1 and 1.2 kb, which reacted with the probe in a label ratio of about 0.5 to 1. In the three inbred lines this ratio was essentially the same.

The multigene family which codes for the mouse major urinary proteins (MUPs) consists of approximately 35 genes. Most of these are members of two different groups, Group 1 and Group 2, which can be distinguished by nucleic acid hybridisation. By screening a Charon 4A library of mouse DNA with probes from the 5'-flanking region of a MUP gene, we have isolated clones that contain both a Group 1 and a Group 2 gene, orientated in a divergent fashion, with 15 kb of DNA between the 5' ends of the genes. We show that this pairwise arrangement is the predominant organisation of MUP genes in the BALB/c genome. We argue that the head-to-head gene pair is the unit both of DNA organisation and of evolution. Taking into account the genes themselves, the intervening 15 kb and the homologous 3'-flanking regions, this unit is approximately 45 kb long. We also show that some MUP genes may be linked in a tail-to-tail fashion with 26-28 kb between the 3' ends of two genes. This suggests that the minimum distance between successive 45-kb units is approximately 7 kb.

Culture conditions which maintain hepatocytes in their in vivo state are not known. This hampers the study of liver gene expression and of direct responses of liver genes to hormonal stimulation. We argued that hepatocytes that were unable to divide might retain in vivo characteristics. We therefore plated mouse (BALB/c) hepatocytes on plastic dishes in medium lacking arginine and measured the levels and transcription rates of six tissue-specific mRNAs over a period of days. Alpha-fetoprotein mRNA began to accumulate at about 48 h of culture, and transcription could sometimes be detected after 72 h. The levels and transcription rates of four mRNAs (albumin, alpha-1-antitrypsin, apolipoprotein A1, and major urinary protein [MUP]) fell sharply. The rate of transcription of transferrin mRNA fell less rapidly, and its level remained high, partly due to its longer half-life. The overall pattern of gene expression in the plated cells did not exactly parallel that of either fetal or regenerating liver. The hepatocytes remained responsive to hormonal stimulation. Insulin and dexamethasone each tended to counteract changes in mRNA levels, for example, preventing the accumulation of alpha-fetoprotein mRNA. The effects of insulin were primarily due to changes in transcription rates. Bovine growth hormone and thyroxine elevated the levels of most of the mRNAs. Many of the effects of these hormones, when added singly, could not be ascribed to changes in transcription. The level of MUP mRNA was strongly affected by added hormones. The mRNA level at 5 days was increased by added insulin, dexamethasone, growth hormone, and thyroxine. In the presence of these three hormones, the decay in the transcription rate of the MUP genes was reduced about 10-fold. We conclude that hepatocytes plated under these nongrowing conditions can provide insights into the hormonal responsiveness of tissue-specific genes.

Hybridization between duck DNA and duck haemoglobin mRNA shows that there is little or no reiteration of the haemoglobin genes

BECAUSE of the large DNA content of the eukaryotic genome1,2 and the existence of reiterated DNA sequences3,4, it has been thought that the DNA sequences which specify protein structure are present in many copies per genome. The idea is not immediately compatible with the simple dominance relationships which alleles at many genetic loci show. A special mechanism has been proposed to overcome this difficulty5, which can also be surmounted in many other ways. From a theoretical standpoint, the question of whether DNA sequences which specify protein structure are reiterated is still open.

In two bacterial species the tandem genes coding for 23S and 16S ribosomal RNA are widely scattered, by contrast with the situation in eukaryotes.

Polyribouridylic acid [poly(U)] labelled with tritium was annealed to chromosomes from Rhynchosciara salivary glands to detect the distribution of A-T rich DNA. A clear pattern of binding was observed to several locations corresponding with structural features of the salivary gland polytene chromosomes. Strong binding also occurred to the cytoplasm of certain testicular cells in Rhynchosciara. These findings are interpreted in terms of the distribution of structural and metabolic sequences rich in polyadenylic acid.

The hybridization of rat liver DNA with complementary RNA has been studied using the membrane technique of Gillespie & Spiegelman (1965). Complementary RNA was synthesized in vitro using Micrococcus RNA polymerase and rat liver DNA. Hybridization was rather rapid, but only about 5% of the total DNA became involved in hybrid formation. The hybrid melted sharply with a mean temperature of 72 °C, only 3 deg. C lower than the mean melting temperature of enzymic DNA-RNA hybrid. RNA complementary to at least half of the total DNA was isolated from a 2:1 enzymic DNA-RNA hybrid. Only about 5% of the DNA hybridized with this complementary RNA under standard annealing conditions, showing that the limited hybridization of rat liver DNA is caused by factors other than limited complementarity of this RNA. It is suggested that the factor responsible is the extreme complexity of mammalian DNA. Mammalian DNA is known to contain a minor fraction which, following denaturation, renatures rapidly compared with the bulk of the DNA. Renaturing rat liver DNA reduced its capacity to hybridize with complementary RNA by two-thirds, showing that it is the rapidly renaturing DNA fraction which hybridizes with the RNA. In confirmation of this, when the rapidly renaturing and slowly renaturing DNA fractions were separated by differential adsorption to hydroxylapatite at 70 °C, preferential hybridization of complementary RNA to the rapidly renaturing DNA fraction was observed. Liver nuclear RNA competed completely against the hybridization of complementary RNA with rat liver DNA. DNA-like RNA from liver, kidney, spleen and brain all competed strongly against complementary RNA. It is concluded that the hybridization of DNA and RNA from higher organisms under the usual conditions permits mainly or only the hybridization of the minor DNA fraction which renatures rapidly. RNA complementary to most of this DMA fraction is found in several different tissues.